Compositions and methods for detecting and/or treating inflammation

ABSTRACT

The present disclosure relates to assays and methods for the detection of renal inflammation by measuring the level of P2Y14 and/or UDP-glucose in a sample from a subject, such as a urine sample. The present disclosure also relates to methods for the treatment of renal inflammation by administering a P2Y14 inhibitor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation Application under 35 U.S.C. § 120 ofU.S. application Ser. No. 15/860,937 filed Jan. 3, 2018, which is aContinuation application under 35 U.S.C. § 120 of U.S. application Ser.No. 15/035,033 filed May 6, 2016, which is a 35 U.S.C. § 371 NationalPhase Entry Application of International Application No.PCT/US2014/064525 filed Nov. 7, 2014, which designates the U.S. andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Nos. 61/962,476 filed Nov. 7, 2013, 61/931,146 filed Jan.24, 2014, and 61/931,184 filed Jan. 24, 2014, the contents of each ofwhich are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 3, 2018, isnamed 030258-080682-PCT_SL.txt and is 29,133 bytes in size.

TECHNICAL FIELD

The present invention relates generally to detection and/or treatmentfor inflammation, particularly renal inflammation.

BACKGROUND

Kidney failure is almost always associated with uncontrolledinflammation. Acute kidney injury (AKI) occurs in two thirds ofintensive care unit patients, 40% of patients after cardiac surgery, and23% of all hospitalized patients. In addition, several factors,including ischemia, nephrotoxins, imaging contrast agents and bacterialendotoxins, contribute to the increased incidence of AKI. AKIcontributes to the progression of chronic kidney disease (CKD) toend-stage renal disease. This is accompanied by longer length of stayand, therefore, generates greater costs. High blood pressure anddiabetes, which afflict millions of people in the US, are the top twoleading causes of CKD. In 2012, 69.53 million cases of CKD were reportedin the six major markets (US, France; Germany, Italy, Spain, and UK),with almost 39 million cases in the US alone. It is estimated that theprevalence of CKD will grow by 1.62% annually, reaching 80.83 millioncases by 2022. If detected early in its progression, kidney disease canbe slowed and the transition to dialysis delayed. However, no earlymarkers of renal inflammation are currently available.

Numerous purinergic receptors are expressed in the kidney, andderegulation of purinergic signaling is associated with severalpathologies, including inflammatory renal diseases, hypertension,chronic kidney disease (CKD), acute kidney injury (AKI), diabeticnephropathy and glomerulonephritis (Arulkumaran, N., et al., FrontPhysiol, 2013, 4:194; Burnstock, G., et al., Purinergic Signal., 2014,10, 71-101). Purinergic receptors are also involved in the regulation ofwater, electrolyte, and volume homeostasis by collecting duct principalcells (Praetorius, H. A., and Leipziger, J., Annu Rev Physiol, 2010,72:377-393; Rieg, T., and Vallon, V., Am J Physiol Regul Integr CompPhysiol, 2009, 296:R419-427; Vallon, V., et al., Wiley Interdiscip RevMembr Transp Signal, 2012, 1:731-742; Kishore, B. K., et al., PurinergicSignal, 2009, 5:491-499). However, very little is known on thepurinergic regulation of the other major cell type of the collectingduct, the intercalated cell (IC). ICs participate in the maintenance ofacid/base homeostasis via the proton-pumping V-ATPase (Breton, S., andBrown, D., Physiology (Bethesda), 2013, 28:318-329; Wagner, C. A., etal., Physiol. Rev., 2004, 84:1263-1314). In the epididymis, ATP andadenosine are potent activators of V-ATPase-dependent proton secretionin clear cells, which are analogous to ICs (Belleannee, C., et al., Am JPhysiol Cell Physiol, 2010, 298:C817-830). Extracellular ATP stimulatesbone resorption in osteoclasts, a process that also requires activity ofthe V-ATPase (Gallagher, J. A. J Musculoskelet Neuronal Interact, 2004,4:125-127; Kaunitz, J. D., and Yamaguchi, D. T., J Cell Biochem, 2008,105:655-662). These studies suggest a role for the purinergic regulationof acid/base transport in the kidney, but the purinergic receptorsignature of ICs still remains to be characterized.

Nucleotide-activated purinergic receptors are separated into twofamilies, P2X receptors that are ligand-gated ion channels, and P2Yreceptors that are G protein-coupled receptors (GPCRs) (Praetorius, H.A., and Leipziger, J., Annu Rev Physiol, 2010, 72:377-393; Rieg, T., andVallon, V., Am J Physiol Regul Integr Comp Physiol, 2009, 296:R419-427;Vallon, V., et al., Wiley Interdiscip Rev Membr Transp Signal, 2012,1:731-742). While p2y5 was initially proposed to be anucleotide-receptor, based on its homology to other P2 receptors, it wassubsequently shown to be insensitive to nucleotides (Li, Q., et al.,Biochem Biophys Res Commun, 1997, 236:455-460), but to instead mediatelysophosphatidic acid (LPA) signaling (Lee, C. W., et al., J Biol Chem,2006, 281:23589-23597). Similarly, p2y10 is a lysophospholipid receptorthat is not activated by nucleotides (Murakami, M., et al., BiochemBiophys Res Commun, 2008, 371:707-712). The P2Y14 receptor (also knownas GPR105) is the most recent member of the P2Y receptor family(Freeman, K., et al., Genomics, 2001, 78:124-128; Chambers, J. K., etal., J Biol Chem, 2000, 275:10767-10771).

There is a clear unmet need for novel strategies aimed at detectingearly signs of inflammation (before the infiltration of pro-inflammatoryimmune cells in the damaged tissue) as well as for the treatment ofinflammation in the kidney.

SUMMARY

The technology disclosed herein is based, in part, on the discovery thatrenal intercalated cells can function as sensors, mediators, andeffectors of inflammation in the kidney via P2Y14, a receptor thatdetects the danger molecule UDP-glucose, which is released from injuredcells. It was further discovered that the expression of P2Y14 waselevated in mice with renal inflammation. The technology disclosedherein can permit the detection of early stage inflammation.

In one aspect, the technology disclosed herein provides an assaycomprising: (i) measuring, in a sample obtained from a subject, a levelof P2Y14; (ii) comparing the level of P2Y14 with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of P2Y14 is above the reference level; and (b) not having renalinflammation if the level of P2Y14 is at or below the reference level.

In one embodiment, when the level of P2Y14 is above the reference level,the method further comprises providing a treatment appropriate fortreating renal inflammation.

In one embodiment, the treatment comprises a P2Y14 inhibitor.

In one embodiment, the level of P2Y14 is a protein level.

In one embodiment, the level of P2Y14 is measured by an immunoassay.

In one embodiment, the sample is contacted with an anti-P2Y14 antibody.

In one embodiment, the anti-P2Y14 antibody is detectably labeled orcapable of generating a detectable signal.

In one embodiment, the antibody is fluorescently labeled.

In one embodiment, the level of P2Y14 is measured by measuring a nucleicacid encoding P2Y14.

In one embodiment, the reference level is an average P2Y14 level in apopulation of healthy subjects.

In one embodiment, the reference level is two standard deviations abovean average P2Y14 level in a population of healthy subjects.

In one embodiment, the assay detects early stage inflammation.

In one embodiment, the assay further comprises measuring a level ofUDP-glucose in a sample obtained from the subject, and comparing thelevel of UDP-glucose with a reference level.

In one embodiment, the sample is a urine sample.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human.

In one aspect, the technology disclosed herein provides a method ofdetecting renal inflammation of a subject, the method comprising: (i)assaying, in a sample obtained from a subject, a level of P2Y14; (ii)comparing the level of P2Y14 with a reference level; and (iii)identifying the subject as (a) having renal inflammation if the level ofP2Y14 is above the reference level; and (b) not having renalinflammation if the level of P2Y14 is at or below the reference level.

In one embodiment, when the level of P2Y14 is above the reference level,the method further comprises providing a treatment appropriate fortreating renal inflammation.

In one embodiment, the treatment comprises a P2Y14 inhibitor.

In one embodiment, the level of P2Y14 is a protein level.

In one embodiment, the level of P2Y14 is measured by an immunoassay.

In one embodiment, the sample is contacted with an anti-P2Y14 antibody.

In one embodiment, the anti-P2Y14 antibody is detectably labeled orcapable of generating a detectable signal.

In one embodiment, the antibody is fluorescently labeled.

In one embodiment, the level of P2Y14 is measured by measuring a nucleicacid encoding P2Y14.

In one embodiment, the reference level is an average P2Y14 level in apopulation of healthy subjects.

In one embodiment, the reference level is two standard deviations abovean average P2Y14 level in a population of healthy subjects.

In one embodiment, the method detects early stage inflammation.

In one embodiment, the method further comprises measuring a level ofUDP-glucose in a sample obtained from the subject, and comparing thelevel of UDP-glucose with a reference level.

In one embodiment, the sample is a urine sample.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human.

In one aspect, the technology disclosed herein provides a method ofmonitoring treatment progress in a subject suffering from renalinflammation, the method comprising: (i) measuring, at a first timepoint, a first level of P2Y14 in a first sample obtained from thesubject; (ii) administering to the subject a therapeutic agent fortreating renal inflammation; and (iii) measuring, at a second timepoint, a second level of P2Y14 in a second sample obtained from thesubject, wherein the second time point is later than the first timepoint and after said administration, and wherein if the second level issignificantly lower than the first level, then the treatment isconsidered to be effective.

In one embodiment, the first sample and the second sample are urinesamples.

In one embodiment, the therapeutic agent is a P2Y14 inhibitor.

In one embodiment, the subject is a human.

In one aspect, the technology disclosed herein provides a method oftreating a subject having renal inflammation, the method comprisingadministering a P2Y14 inhibitor to the subject.

In one embodiment, the P2Y14 inhibitor is PPTN or an anti-P2Y14antibody.

In one embodiment, the subject is a human.

In one aspect, the technology disclosed herein provides a method oftreating a subject determined to have a level of P2Y14 above a referencelevel, the method comprising administering a treatment appropriate fortreating renal inflammation.

In one embodiment, the treatment appropriate for treating renalinflammation comprises a P2Y14 inhibitor.

In one embodiment, the P2Y14 inhibitor is PPTN or an anti-P2Y14antibody.

In one embodiment, the subject is a human.

In one aspect, the technology disclosed herein provides an assaycomprising: (i) measuring, in a sample obtained from a subject, a levelof UDP-glucose; (ii) comparing the level of UDP-glucose with a referencelevel; and (iii) identifying the subject as (a) having renalinflammation if the level of UDP-glucose is above a reference level; and(b) not having renal inflammation if the level of UDP-glucose is at orbelow the reference level.

In one embodiment, when the level of UDP-glucose is above the referencelevel, the method further comprises providing a treatment appropriatefor treating renal inflammation.

In one embodiment, the treatment comprises a P2Y14 inhibitor.

In one embodiment, the reference level is an average UDP-glucose levelin a population of healthy subjects.

In one embodiment, the reference level is two standard deviations abovean average UDP-glucose level in a population of healthy subjects.

In one embodiment, the assay detects early stage inflammation.

In one embodiment, the sample is a urine sample.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human.

In one aspect, the technology disclosed herein provides a method ofdetecting renal inflammation of a subject, the method comprising: (i)assaying, in a sample obtained from a subject, a level of UDP-glucose;(ii) comparing the level of UDP-glucose with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of UDP-glucose is above a reference level; and (b) not havingrenal inflammation if the level of UDP-glucose is at or below thereference level.

In one embodiment, when the level of UDP-glucose is above the referencelevel, the method further comprises providing a treatment appropriatefor treating renal inflammation.

In one embodiment, the treatment comprises a P2Y14 inhibitor.

In one embodiment, the reference level is an average UDP-glucose levelin a population of healthy subjects.

In one embodiment, the reference level is two standard deviations abovean average UDP-glucose level in a population of healthy subjects.

In one embodiment, the method detects early stage inflammation.

In one embodiment, the sample is a urine sample.

In one embodiment, the subject is a mammal.

In one embodiment, the mammal is a human.

In one aspect, the technology disclosed herein provides a method ofmonitoring treatment progress in a subject suffering from renalinflammation, the method comprising: (i) measuring, at a first timepoint, a first level of UDP-glucose in a first sample obtained from thesubject; (ii) administering to the subject a therapeutic agent fortreating renal inflammation; and (iii) measuring, at a second timepoint, a second level of UDP-glucose in a second sample obtained fromthe subject, wherein the second time point is later than the first timepoint and after said administration, and wherein if the second level issignificantly lower than the first level, then the treatment isconsidered to be effective.

In one embodiment, the first sample and the second sample are urinesamples.

In one embodiment, the therapeutic agent is a P2Y14 inhibitor.

In one embodiment, the subject is a human.

In one aspect, the technology disclosed herein provides a method oftreating a subject determined to have a level of UDP-glucose above areference level, the method comprising administering a treatmentappropriate for treating renal inflammation.

In one embodiment, the treatment appropriate for treating renalinflammation comprises a P2Y14 inhibitor.

In one embodiment, the P2Y14 inhibitor is PPTN or an anti-P2Y14antibody.

In one embodiment, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is experimental data showing RT-PCR detection of P2 receptors inwhole kidney (lower panel) and in EGFP(+) cells (upper panel) isolatedby fluorescence-activated cell sorting (FACS) from B1-EGFP mousekidneys. GAPDH was used as positive control. NC=no template control.

FIG. 2A is a bar graph showing quantitative PCR detection of IC markers(B1, AE1, pendrin,) in EGFP(+) vs EGFP(−) cells isolated from renalcortex and medulla. Values are normalized to GAPDH and represented asfold changes relative to the values obtained in cortical EGFP(+) cells.

FIG. 2B is a bar graph showing relative P2 receptor mRNA levels,analyzed by quantitative PCR in medullary and cortical EGFP(+) cells.Data are normalized for GAPDH and are expressed as mean±SEM (n=3),*P<0.05, **P<0.001.

FIG. 3A-FIG. 3D are images showing immunofluorescence localization ofP2Y₁₄ in mouse kidney. Cortical (FIG. 3A) and medullary (FIG. 3B)sections double-labeled for P2Y₁₄ (visualized as green) and the V-ATPaseB1 subunit (visualized as red). P2Y₁₄ was detected in intercalated cells(ICs) identified by their positive labeling for the V-ATPase (visualizedas yellow in the merge panel shown in FIG. 3A). No P2Y₁₄ was detected indistal tubule cells, which also express the V-ATPase (visualized as redin the merge panel shown in FIG. 3A). The P2Y₁₄ staining was abolishedafter pre-incubation of the P2Y₁₄ antibody with its immunizing peptidein the cortex (FIG. 3C) and medulla (FIG. 3D). Scale bars=25 μm.

FIG. 4A and FIG. 4B are experimental data showing the expression ofP2Y₁₄ in EGFP(+) cells.

FIG. 4A shows representative immunoblot profile of P2Y14 in two EGFP(+)cell samples isolated by FACS.

FIG. 4B shows binding of [3H]UDP-glucose to total membranes preparedfrom FACS isolated EGFP(+) and EGFP(−) cells in the presence or absenceof a saturating concentration (10⁻⁵ M) of unlabeled UDP-glucose or ATP.Data are represented as fold changes compared to the binding measured inthe presence of unlabeled UDP-glucose. Each bar represents the averageof 3 independent experiments performed in triplicate. Values areexpressed as mean±SEM, *P<0.05.

FIG. 5A and FIG. 5B are experimental data showing the effect ofUDP-glucose on ERK1/2 phosphorylation in FACS isolated EGFP(+) andEGFP(−) cells.

FIG. 5A is a set of representative immunoblots of pERK (upper lanes) andtotal ERK (lower lanes) in EGFP(+) cells (left panel) and EGFP(−) cells(right panel).

FIG. 5B is a bar graph showing that densitometry analysis of ERKphosphorylation is represented as % increase of the ratio of pERK/totalERK relative to control. Values (in % of control) are means±SEM (n=3),*P<0.05.

FIG. 6A-FIG. 6F are experimental data showing P2Y14 expression inMDCK-C11 cells.

FIG. 6A shows RT-PCR analysis of IC markers including the V-ATPase a4subunit (V0A4), the V-ATPase B1 subunit (V1B1) and AE1, and theprincipal cell marker aquaporin 2 (AQP2), as well as P2Y₁₄ in MDCK-C11cells.

FIG. 6B shows representative immunoblots following plasma membranebiotinylation showing cell surface versus total protein expression ofthe V-ATPase B1 and A subunits, and actin.

FIG. 6C shows RT-PCR detection of P2 receptors in MDCK-C11 cells.

FIG. 6D shows immunoblot profile of P2Y₁₄ expression in MDCK-C11. Plasmamembrane (left) and total cell expression (right) are represented undercontrol conditions (C) and after treatment with endoglycosydase H (H)and PNGase F (F).

FIG. 6E shows X-Z confocal microscopy representation of MDCK-C11 cellsgrown on filter, showing P2Y₁₄ expression (visualized as red). Plasmamembrane is labeled with biotin-streptavidin FITC (visualized as green).The merge panel shows partial co-localization of P2Y₁₄ with biotin inthe apical membrane (visualized as orange/yellow) as well as sub-apicallocalization (visualized as red). Scale bars=4 μm.

FIG. 6F shows concentration-dependent inhibition of [³H]UDP-glucosebinding to MDCK-C11 membranes by unlabeled ligands. Membranes (15 μgprotein) were incubated for 3 hours at 22° C. with [³H]UDP-glucose (3nM) and increasing concentrations of UDP-glucose or ATP. Each pointrepresents the average of 4 independent experiments performed intriplicate. The data are expressed as values relative to the totalbinding observed in the absence of unlabeled ligand and are correctedfor non specific binding determined in the presence of a saturatingconcentration of UDP-glucose (10 μM).

FIG. 7A and FIG. 7B are experimental data showing P2Y₁₄ activation byUDP-glucose increases ERK1/2-phosphorylation in MDCK-C11 cells.

FIG. 7A shows representative immunoblots showing triplicates of ERK1/2phosphorylation (upper lane) versus total ERK1/2 (lower lane) in cellspretreated with vehicle or the P2Y₁₄ antagonist PPTN (10 in the absence(CTRL) or presence of 100 μM UDP-glucose (UDP-glu).

FIG. 7B shows quantification of the ratio of p-ERK/total ERK (lowerpanel) showed that PPTN prevented the increase in ERK1/2 phosphorylationinduced by UDP-glu in cells treated with the vehicle only. Values arerepresented as % of controls as means±SEM (n=3), **p<0.001.

FIG. 8A shows quantitative PCR detection of pro-inflammatory mediatorsin MDCK-C11 cells under control conditions and 4 h after 100 μMUDP-glucose treatment. Data are represented as % changes relative tocontrol. All values are normalized to GAPDH and are shown as Means±SEM(n=5), **P<0.001.

FIG. 8B shows quantification of changes in IL-8 (left) and CCL2 (right)mRNA expression in MDCK-C11 cells pretreated with the vehicle only orwith the MEK inhibitor, PD98059 (50 μM) for 30 minutes in the absence(CTRL) or presence of 100 tM UDP-glucose (UDP-glu).

FIG. 8C shows quantification of changes in IL-8 (left) and CCL2 (right)mRNA expression in MDCK-C11 cells pretreated with the vehicle only orwith PPTN (10 tM) for 30 minutes, in the absence (CTRL) or presence of100 tM UDP-glucose (UDP-glu). Data are represented as % changes relativeto control. Values are means±SEM (n=3), *P<0.05, **P<0.001.

FIG. 9 shows quantitative PCR detection of pro-inflammatory mediators inEGFP(+) cells isolated by FACS from B1-EGFP mice 4 h after injectionwith saline (sham) or with saline containing 100 μM UDP-glucose(UDP-glu). All values are normalized to GAPDH. Data are represented as %changes relative to control. Values are mean±SEM (n=4), *P<0.05,**P<0.001.

FIG. 10A and FIG. 10B show flow cytometry analysis of immune cellinfiltration in the kidneys of mice 48 h after injection with saline(sham) or 100 μM UDP-glucose (UDP-glu). Changes in kidney medulla (FIG.10A) or cortex (FIG. 10B) infiltrated immune cell counts are representedas % changes relative to control. Values are means of percent of eachcell population ±SEM from 4-6 animals. *P<0.05.

FIG. 11A-FIG. 11E are experimental data showing immunolocalization ofneutrophils in kidney medulla of mice 48 h after injection with saline(Sham) or 100 tM UDP-glucose.

FIG. 11A and FIG. 11B are a set of mosaic, low magnification images ofkidney medulla double-labeled for P2Y₁₄ (visualized as green) and theneutrophil marker Ly6G (visualized as red; white circles) from miceinjected with saline (FIG. 11A) or 100 tM UDP-glucose (FIG. 11B). Scalebars=200 μm.

FIG. 11C-FIG. 11D″ is a set of high magnification images of themedullary regions showing neutrophil infiltration after UDP-glucoseinjection (FIG. 11D, FIG. 11D′, FIG. 11D″) but not in the sham animals(FIG. 11C, FIG. 11C′, FIG. 11C″). Neutrophils (arrows) were often seenin close proximity to ICs, labeled for P2Y₁₄ (visualized as green),after UDP-glucose injection. Scale bars=25 μm.

FIG. 11E is a bar graph showing flow cytometry analysis of neutrophilcounts (Ly6G positive) from cortex and medulla in control mice (Sham)and after UDP-glucose injection.

FIG. 12 is a set of images showing that P2Y14 is expressed in humankidney intercalated cells.

FIG. 13 is a bar graph showing that UDG-glucose increases ERKphosphorylation in ICs isolated by FACS.

FIG. 14 is a bar graph showing that PPTN inhibits UDP-glucose dependentERK phosphorylation in ICs isolated by FACS.

DETAILED DESCRIPTION

The present disclosure is based, in part, on the discovery that P2Y14and/or UDG-glucose can be used as biomarkers for the detection of renalinflammation. Detection of elevated levels of P2Y14 and/or UDG-glucosein a sample from a subject compared to a reference level can thus beused to indicate the presence of renal inflammation in the subject.Advantageously, early stage inflammation can be detected using one orboth of these biomarkers. Accordingly, embodiments of the technologydescribed herein provide, among other things, assays, methods, andsystems for determining whether a subject has renal inflammation,methods for monitoring treatment progress in a subject having renalinflammation, and methods for treating a subject having renalinflammation. It should be noted that P2Y14 and UDP-glucose can be usedas biomarkers independently or in combination.

The P2Y₁₄ receptor (also known as GPR105 or SC-GPR) is the most recentmember of the P2Y receptor family (Freeman, K., et al., Genomics, 2001,78:124-128; Chambers, J. K., et al., J Biol Chem, 2000,275:10767-10771). P2Y14 is specifically activated by nucleotide sugarsincluding UDP-glucose and it is insensitive to ADP/ATP and UTP(Chambers, J. K., et al., J Biol Chem, 2000, 275:10767-10771). Mostnucleotides are rapidly degraded after their release, but UDP-glucoseresists hydrolysis by ectonucleotidases (Zimmermann, H., NaunynSchmiedebergs Arch Pharmacol, 2000, 362:299-309). While virtually allcells release nucleotides under basal conditions (Lazarowski, E. R., etal., Mol Pharmacol, 2003, 63:1190-1197), this release can be accentuatedin response to stimuli leading to activation of purinergic receptors(Leipziger, J., Curr Opin Nephrol Hypertens, 2011, 20:518-522).UDP-glucose, extracellular ATP and adenosine are emerging asimmune-regulatory factors known as DAMPs (damage associated molecularpattern) molecules (Chen, Y., et al., Science, 2006, 314:1792-1795;Elliott, M. R., et al., Nature, 2009, 461:282-286). DAMPs initiatesterile inflammatory reactions, as opposed to PAMP (pathogen associatedmolecular patterns), which perpetuate infectious pro-inflammatoryresponses (Elliott, M. R., et al., Nature, 2009, 461:282-286; Harden, T.K., et al., Acta Physiol (Oxf), 2010, 199:149-160; Chen, G. Y., andNunez, G. Nat Rev Immunol, 2010, 10:826-837; Kono, H., and Rock, K. L.,Nat Rev Immunol, 2008, 8:279-289).

As used herein, the term “P2Y14” generally refers to a P2Y14 polypeptideor a P2Y14 polynucleotide that is similar or identical to the sequenceof a wild-type P2Y14.

The wild-type P2Y14 sequences of various species are available on theworld wide web such as the NCBI. For example, human P2Y14 amino acidsequence is included herein as (SEQ ID NO: 115) for reference:

P2Y purinoceptor 14 (P2Y14) [Homo sapiens] (NCBI Reference Sequence: NP001074924.1):1 minststqpp descsqnlli tqqiipvlyc mvfiagilln gvsgwiffyv pssksfiiyl61 kniviadfvm sltfpfkilg dsglgpwqln vfvcrvsavl fyvnmyvsiv ffglisfdry121 ykivkplwts fiqsysyskl lsvivwmlml llavpniilt nqsvrevtqi kcielkselg181 rkwhkasnyi fvaifwivfl llivfytait kkifkshlks srnstsvkkk ssrnifsivf241 vffvcfvpyh iaripytksq teahyscqsk eilrymkeft lllsaanvcl dpiiyfflcq301 pfreilckkl hiplkaqndl disrikrgnt tlestdtl (SEQ ID NO: 115)

In one aspect, the technology disclosed herein provides a method ofdetecting renal inflammation of a subject, the method comprising: (i)assaying, in a sample obtained from a subject, a level of P2Y14; (ii)comparing the level of P2Y14 with a reference level; and (iii)identifying the subject as (a) having renal inflammation if the level ofP2Y14 is above the reference level; and (b) not having renalinflammation if the level of P2Y14 is at or below the reference level.

In various aspects described herein, methods for measuring P2Y14 or afragment thereof from a sample are known in the art, including, but notlimited to measuring mRNA expression using PCR or real-time PCR, proteinanalysis using western blot, immunoassay, and/or sequencing analysis.Thus, in some embodiments, nucleic acid molecules from a patient'ssample can be isolated or assayed to measure P2Y14 mRNA expression, orproteins can be isolated or assayed to measure P2Y14 protein expression.

P2Y14 protein or polypeptide levels can be measured using a variety ofmethods known in the art including, but not limited to, an immunologicaltest, surface plasmon resonance, and photonic crystal-based detection.Immunological tests include, for example, competitive andnon-competitive assay systems using techniques such as Western blots,radioimmunoassay (MA), ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusionassays, agglutination assays, e.g. latex agglutination,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, e.g. FIA (fluorescence-linked immunoassay),chemiluminescence immunoassays (CLIA), electrochemiluminescenceimmunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests orimmunoassay (LFIA), magnetic immunoassay (MIA), and protein Aimmunoassays. Methods for performing such assays are known in the art,provided an appropriate antibody reagent is available. In someembodiments, the immunoassay can be a quantitative or asemi-quantitative immunoassay.

An immunoassay is a biochemical test that measures the concentration ofa substance in a biological sample, typically a fluid sample, using theinteraction of an antibody or antibodies to its antigen. The assay takesadvantage of the highly specific binding of an antibody with itsantigen. In some embodiments, specific binding of the P2Y14 moleculewith an anti-P2Y14 antibody forms a P2Y14-antibody complex. The complexcan then be detected by a variety of methods known in the art. Animmunoassay also often involves the use of a detection antibody.Anti-P2Y14 antibodies are commercially available through vendors such asAlomone Labs (Jerusalem, Isarel), Sigma Aldrich, and Merck Millipore. Inone embodiment, the anti-P2Y14 antibody is detectably labeled or capableof generating a detectable signal. In one embodiment, the anti-P2Y14antibody is fluorescently labeled.

In some embodiments, P2Y14 levels are measured by ELISA, also calledenzyme immunoassay or EIA. ELISA is a biochemical technique that detectsthe presence of an antibody or an antigen in a sample.

In one embodiment, an ELISA involving at least one antibody withspecificity for the particular desired antigen (i.e. P2Y14) can beperformed. A known amount of sample and/or antigen is immobilized on asolid support (usually a polystyrene micro titer plate). Immobilizationcan be either non-specific (e.g., by adsorption to the surface) orspecific (e.g. where another antibody immobilized on the surface is usedto capture antigen or a primary antibody). After the antigen isimmobilized, the detection antibody is added, forming a complex with theantigen. The detection antibody can be covalently linked to an enzyme,or can itself be detected by a secondary antibody which is linked to anenzyme through bio-conjugation. Between each step the plate is typicallywashed with a mild detergent solution to remove any proteins orantibodies that are not specifically bound. After the final wash stepthe plate is developed by adding an enzymatic substrate to produce avisible signal, which indicates the quantity of antigen in the sample.Older ELISAs utilize chromogenic substrates, though newer assays employfluorogenic substrates with much higher sensitivity.

In one embodiment, a sandwich ELISA is used, where two types ofanti-P2Y14 antibodies can be used. There are other different forms ofELISA, which are well known to those skilled in the art. Standardtechniques known in the art for ELISA are described in “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904.These references are hereby incorporated by reference for theirteachings on ELISA.

In one embodiment, the levels of P2Y14 in a sample can be detected by alateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test. LFIAs are a simple deviceintended to detect the presence (or absence) of antigen, e.g. P2Y14, ina fluid sample. There are currently many LFIA tests used for medicaldiagnostics either for home testing, point of care testing, orlaboratory use. LFIA tests are a form of immunoassay in which the testsample flows along a solid substrate via capillary action. After thesample is applied to the test strip it encounters a colored reagent(generally comprising antibody specific for the test target antigen)bound to microparticles which mixes with the sample and transits thesubstrate encountering lines or zones which have been pretreated withanother antibody or antigen. Depending upon the level of P2Y14 presentin the sample the colored reagent can be captured and become bound atthe test line or zone. LFIAs are essentially immunoassays adapted tooperate along a single axis to suit the test strip format or a dipstickformat. Strip tests are extremely versatile and can be easily modifiedby one skilled in the art for detecting an enormous range of antigensfrom fluid samples such as urine, blood, water samples etc. Strip testsare also known as dip stick test, the name bearing from the literalaction of “dipping” the test strip into a fluid sample to be tested.LFIA strip tests are easy to use, require minimum training and caneasily be included as components of point-of-care test (POCT)diagnostics to be use on site in the field. LFIA tests can be operatedas either competitive or sandwich assays. Sandwich LFIAs are similar tosandwich ELISA. The sample first encounters colored particles which arelabeled with antibodies raised to the target antigen. The test line willalso contain antibodies to the same target, although it may bind to adifferent epitope on the antigen. The test line will show as a coloredband in positive samples. In some embodiments, the lateral flowimmunoassay can be a double antibody sandwich assay, a competitiveassay, a quantitative assay or variations thereof. Competitive LFIAs aresimilar to competitive ELISA. The sample first encounters coloredparticles which are labeled with the target antigen or an analogue. Thetest line contains antibodies to the target/its analogue. Unlabelledantigen in the sample will block the binding sites on the antibodiespreventing uptake of the colored particles. The test line will show as acolored band in negative samples. There are a number of variations onlateral flow technology. It is also possible to apply multiple capturezones to create a multiplex test.

A typical test strip consists of the following components: (1) sampleapplication area comprising an absorbent pad (i.e., the matrix ormaterial) onto which the test sample is applied; (2) conjugate orreagent pad-this contains antibody reagent(s) specific to the targetantigen (e.g. specific P2Y14 or fragment thereof) which can beconjugated to colored particles (e.g., colloidal gold particles, orlatex microspheres); (3) test results area comprising a reactionmembrane—typically a hydrophobic nitrocellulose or cellulose acetatemembrane onto which antibody reagents (e.g., anti-P2Y14 antibodies) areimmobilized in a line across the membrane as a capture zone or test line(a control zone may also be present, containing antibodies specific forthe antibody reagents conjugated to the particles or microspheres); and(4) optional wick or waste reservoir—a further absorbent pad designed todraw the sample across the reaction membrane by capillary action andcollect it. The components of the strip are usually fixed to an inertbacking material and may be presented in a simple dipstick format orwithin a plastic casing with a sample port and reaction window showingthe capture and control zones. While not strictly necessary, most testswill incorporate a second line which contains an antibody that picks upfree latex/gold in order to confirm the test has operated correctly.

The use of “dip sticks” or LFIA test strips and other solid supportshave been described in the art in the context of an immunoassay for anumber of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982;6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser.No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082,which are incorporated herein by reference in their entirety, arenon-limiting examples of such lateral flow test devices. Examples ofpatents that describe the use of “dip stick” technology to detectsoluble antigens via immunochemical assays include, but are not limitedto U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which areincorporated by reference herein in their entireties. The apparatusesand methods of these three patents broadly describe a first componentfixed to a solid surface on a “dip stick” which is exposed to a solutioncontaining a soluble antigen that binds to the component fixed upon the“dip stick,” prior to detection of the component-antigen complex uponthe stick. It is within the skill of one in the art to modify theteachings of this “dip stick” technology for the detection of P2Y14using antibody reagents as described herein.

A urine dipstick is a colorimetric chemical assay. It consists of areagent stick-pad, which is immersed in a fresh urine specimen and thenwithdrawn. After predetermined times the colors of the reagent pad arecompared to standardized reference charts. The urine dipstick offers aninexpensive and fast method to perform screening urinalyses, which helpin identifying the presence of various diseases or health problems. Aurine dipstick provides a simple and clear diagnostic guideline and canbe used in the methods and kits as described herein. Accordingly, oneaspect of the present technology relates to a method for detecting P2Y14using a device, such as a dipstick, as described herein.

Other techniques can be used to detect the level of P2Y14 in a sample.One such technique is the dot blot, and adaptation of Western blotting(Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Westernblot, P2Y14 can be dissociated with detergents and heat, and separatedon an SDS-PAGE gel before being transferred to a solid support, such asa nitrocellulose or PVDF membrane. The membrane is incubated with anantibody reagent specific for P2Y14. The membrane is then washed toremove unbound proteins and proteins with non-specific binding.Detectably labeled enzyme-linked secondary or detection antibodies canthen be used to detect and assess the amount of P2Y14 in the sampletested. The intensity of the signal from the detectable labelcorresponds to the amount of enzyme present, and therefore the amount ofP2Y14. Levels can be quantified, for example by densitometry.

The level of P2Y14 can also be measured by measuring the biologicalactivity of P2Y14, such as the downstream signaling activity of P2Y14,or the UDP-glucose binding activity.

In some embodiments, the level of P2Y14 can also be measured bymeasuring the level of mRNA encoding P2Y14. Methods of measuring mRNAlevels include, but are not limited to, polymerase chain reaction (PCR,e.g., quantitative or semi-quantitative, or reverse transcription-PCR),hybridization assay, Northern blotting, primer extension, ribonucleaseprotection, and variations of these.

Injured cells release UDP-glucose, a damage-associated molecular patternmolecule (DAMP) that signals through the purinergic receptor P2Y14 toinitiate the release of pro-inflammatory chemokines (PIC). And thusUDP-glucose level can also be used as an indicator for inflammation. Inone aspect, the technology disclosed herein provides a method ofdetecting renal inflammation of a subject, the method comprising: (i)assaying, in a sample obtained from a subject, a level of UDP-glucose;(ii) comparing the level of UDP-glucose with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of UDP-glucose is above a reference level; and (b) not havingrenal inflammation if the level of UDP-glucose is at or below thereference level.

UDP-glucose levels can be measured using a variety of methods known inthe art, such as HPLC. In some embodiments, UDP-glucose levels can bemeasured using the protocols described in Barrett et al., Molec.Pharmacol., 2013, 84, 41-49, the contents of which are incorporatedherein by reference in their entirety.

P2Y14 and/or UDP-glucose can be used for monitoring the treatmentprogress in a subject having renal inflammation. For example, bymeasuring the levels of P2Y14 and/or UDP-glucose at multiple time pointsduring the treatment, a decrease in the levels of P2Y14 and/orUDP-glucose over time can indicate that the treatment is effective. Inone aspect, the technology disclosed herein provides a method ofmonitoring treatment progress in a subject suffering from renalinflammation, the method comprising: (i) measuring, at a first timepoint, a first level of P2Y14 or UDP-glucose in a first sample obtainedfrom the subject; (ii) administering to the subject a therapeutic agentfor treating renal inflammation; and (iii) measuring, at a second timepoint, a second level of P2Y14 or UDP-glucose in a second sampleobtained from the subject, wherein the second time point is later thanthe first time point and after said administration, and wherein if thesecond level is significantly lower than the first level, then thetreatment is considered to be effective.

Sample

The terms “sample”, “biological sample”, or “test sample” as used hereindenote a sample taken or isolated from a biological organism, e.g., ananimal or human. Exemplary biological samples include, but are notlimited to, a biofluid sample; a body fluid sample, blood (includingwhole blood); serum; plasma; urine; saliva; a biopsy and/or tissuesample etc. The term also includes a mixture of the above-mentionedsamples. The term “sample” also includes untreated or pretreated (orpre-processed) biological samples. In some embodiments, a sample cancomprise one or more cells from the subject. In some embodiments, thesample used for the assays and methods described herein can comprise aurine sample collected from a subject to be tested. In some embodiments,the sample used for the assays and methods described herein can comprisea blood sample collected from a subject to be tested.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using previously isolated samples (e.g.isolated at a prior time point and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected sample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. In some embodiments, a test sample canbe a pre-processed test sample, for example, supernatant or filtrateresulting from a treatment selected from the group consisting ofcentrifugation, filtration, thawing, purification, and any combinationsthereof. In some embodiments, the test sample can be treated with achemical and/or biological reagent. Chemical and/or biological reagentscan be employed to protect and/or maintain the stability of the sample,including biomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. In some embodiments, the test sample can be a frozen testsample, e.g., a frozen tissue. The frozen sample can be thawed beforeemploying methods, assays and systems described herein. After thawing, afrozen sample can be centrifuged before being subjected to methods,assays and systems described herein. In some embodiments, the testsample is a clarified test sample, for example, by centrifugation andcollection of a supernatant comprising the clarified test sample.

Reference Level

In some embodiments, the reference level can correspond to an averagelevel of P2Y14 or UDP-glucose in a sample (e.g., urine) of a normalhealthy subject or a population of normal healthy subjects. This wouldbe a “normal” level. As used herein, the term “normal healthy subject”refers to a subject who has no symptoms of any diseases or disorders, orwho is not identified with any diseases or disorders, or who is not onany medication treatment, or a subject who is identified as healthy byphysicians based on medical examinations. It should be noted and obviousto a skilled artisan that one should only compare the measured P2Y14level with the reference level of P2Y14, and the measured UDP-glucoselevel with the reference level of UDP-glucose.

In some embodiments, the reference level can be at least one standarddeviation (including, e.g., at least two standard deviations) above theaverage level of P2Y14 or UDP-glucose in a sample (e.g., urine) of anormal healthy subject or a population of normal healthy subjects. Insome embodiments, the reference level can be at least two standarddeviations above the average level of P2Y14 or UDP-glucose in a sample(e.g., urine) of a normal healthy subject or a population of normalhealthy subjects. In these embodiments, any level above the referencelevel is considered to be significantly different from the average levelof P2Y14 or UDP-glucose in a sample of a normal healthy subject or apopulation of normal healthy subjects.

In some embodiments, the reference level can be a level of P2Y14 orUDP-glucose in a control sample, a pooled sample of control individuals,or a numeric value or range of values based on the same. It is alsocontemplated that a set of standards can be established with referencelevels providing thresholds indicative of the severity of renalinflammation.

In some embodiments, the reference level can be an average level ofP2Y14 or UDP-glucose in a sample (e.g., urine) of subject or apopulation of subjects without inflammation, or more specifically, renalinflammation. These subjects might have a disease other than renalinflammation. In some embodiments, the reference level can be at leastone standard deviation (including, e.g., at least two standarddeviations) above the average level of P2Y14 or UDP-glucose in a sample(e.g., urine) of a subject or a population of subjects withoutinflammation, or more specifically, renal inflammation.

In some embodiments, the reference level can be a level of P2Y14 orUDP-glucose in a sample (e.g., urine) of the same subject measured at anearlier time point, e.g., before or during the treatment. In theseembodiments, a physician monitors the subject's P2Y14 or UDP-glucoselevels over time for, e.g., determining treatment efficacy or managingthe disease progression.

In some embodiments, the level of P2Y14 measured in a sample from asubject identified as having renal inflammation can be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, at least 200%, or at least 300% higher than the referencelevel.

In some embodiments, the level of UDP-glucose measured in a sample froma subject identified as having renal inflammation can be at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, at least 200%, or at least 300% higher than the referencelevel.

It should be noted that the reference level can be different, dependingon factors such as the sample type from which the reference level isderived, gender, age, weight, and ethnicity. Thus, reference levelsaccounting for these and other variables can provide added accuracy forthe methods described herein.

Computer Systems

In some embodiments of the assays and/or methods described herein, theassay/method comprises or consists essentially of a system fordetermining (e.g. transforming and measuring) the level of P2Y14 and/orUDP-glucose as described herein and comparing it to a reference level.If the comparison system, which can be a computer implemented system,indicates that the amount of the measured level of P2Y14 and/orUDP-glucose is statistically higher than that of the reference amount,the subject from which the sample is collected can be identified ashaving renal inflammation.

In one embodiment, provided herein is a system comprising: (a) at leastone memory containing at least one computer program adapted to controlthe operation of the computer system to implement a method that includes(i) a determination module configured to measure the level of P2Y14and/or UDP-glucose in a test sample obtained from a subject; (ii) astorage module configured to store output data from the determinationmodule; (iii) a computing module adapted to identify from the outputdata whether the measured level of P2Y14 and/or UDP-glucose in the testsample obtained from the subject is higher, by a statisticallysignificant amount, than a reference level, and to provide a retrievedcontent; (iv) a display module for displaying for retrieved content(e.g., the amount of the measured level of P2Y14 and/or UDP-glucose, orwhether the measured level of P2Y14 and/or UDP-glucose is higher thanthe reference level); and (b) at least one processor for executing thecomputer program.

Embodiments can be described through functional modules, which aredefined by computer executable instructions recorded on computerreadable media and which cause a computer to perform method steps whenexecuted. The modules are segregated by function for the sake ofclarity. However, it should be understood that the modules/systems neednot correspond to discreet blocks of code and the described functionscan be carried out by the execution of various code portions stored onvarious media and executed at various times. Furthermore, it should beappreciated that the modules can perform other functions, thus themodules are not limited to having any particular functions or set offunctions.

The computer readable storage media can be any available tangible mediathat can be accessed by a computer. Computer readable storage mediaincludes volatile and nonvolatile, removable and non-removable tangiblemedia implemented in any method or technology for storage of informationsuch as computer readable instructions, data structures, program modulesor other data. Computer readable storage media includes, but is notlimited to, RAM (random access memory), ROM (read only memory), EPROM(erasable programmable read only memory), EEPROM (electrically erasableprogrammable read only memory), flash memory or other memory technology,CD-ROM (compact disc read only memory), DVDs (digital versatile disks)or other optical storage media, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage media, other types ofvolatile and non-volatile memory, and any other tangible medium whichcan be used to store the desired information and which can accessed by acomputer including and any suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programsthat, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable storage medium describedherein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the technology discussed herein. In addition,it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the technology describedherein. The computer executable instructions may be written in asuitable computer language or combination of several languages. Basiccomputational biology methods are known to those of ordinary skill inthe art and are described in, for example, Setubal and Meidanis et al.,Introduction to Computational Biology Methods (PWS Publishing Company,Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods inMolecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

The functional modules of certain embodiments can include at minimum adetermination module, a storage module, a computing module, and adisplay module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The determination module has computer executable instructions to providee.g., levels of expression products etc in computer readable form.

The determination module can comprise any system for detecting a signalresulting from the detection of P2Y14 and/or UDP-glucose in a biologicalsample. In some embodiments, such systems can include an instrument,e.g., a plate reader for measuring absorbance. In some embodiments, suchsystems can include an instrument, e.g., the Cell Biosciences NANOPRO1000™ System (Protein Simple; Santa Clara, Calif.) for quantitativemeasurement of proteins.

The information determined in the determination system can be read bythe storage module. As used herein the “storage module” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the technology describedherein include stand-alone computing apparatus, data telecommunicationsnetworks, including local area networks (LAN), wide area networks (WAN),Internet, Intranet, and Extranet, and local and distributed computerprocessing systems. Storage modules also include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedia, magnetic tape, optical storage media such as CD-ROM, DVD,electronic storage media such as RAM, ROM, EPROM, EEPROM and the like,general hard disks and hybrids of these categories such asmagnetic/optical storage media. The storage module is adapted orconfigured for having recorded thereon, for example, sample name,patient name, and numerical value of the level of P2Y14 and/orUDP-glucose. Such information may be provided in digital form that canbe transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage module. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising expression level information.

In one embodiment of any of the systems described herein, the storagemodule stores the output data from the determination module. Inadditional embodiments, the storage module stores the referenceinformation such as levels of P2Y14 and/or UDP-glucose in healthysubjects. In some embodiments, the storage module stores the informationsuch as levels of P2Y14 and/or UDP-glucose measured from the samesubject in earlier time points.

The “computing module” can use a variety of available software programsand formats for computing the levels of P2Y14 and/or UDP-glucose. Suchalgorithms are well established in the art. A skilled artisan is readilyable to determine the appropriate algorithms based on the size andquality of the sample and type of data. The data analysis can beimplemented in the computing module. In one embodiment, the computingmodule further comprises a comparison module, which compares the levelof P2Y14 and/or UDP-glucose in the test sample obtained from a subjectas described herein with the reference level. By way of example, whenthe level of P2Y14 and/or UDP-glucose in the test sample obtained from asubject is measured, a comparison module can compare or match the outputdata, e.g. with the reference level. In certain embodiments, thereference level has been pre-stored in the storage module. During thecomparison or matching process, the comparison module can determinewhether the level of P2Y14 and/or UDP-glucose in the test sampleobtained from a subject is higher than the reference level to astatistically significant degree. In various embodiments, the comparisonmodule can be configured using existing commercially-available orfreely-available software for comparison purpose, and may be optimizedfor particular data comparisons that are conducted.

The computing and/or comparison module, or any other module, can includean operating system (e.g., UNIX) on which runs a relational databasemanagement system, a World Wide Web application, and a World Wide Webserver. World Wide Web application includes the executable codenecessary for generation of database language statements (e.g.,Structured Query Language (SQL) statements). Generally, the executableswill include embedded SQL statements. In addition, the World Wide Webapplication may include a configuration file which contains pointers andaddresses to the various software entities that comprise the server aswell as the various external and internal databases which must beaccessed to service user requests. The Configuration file also directsrequests for server resources to the appropriate hardware, as may benecessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in aparticular preferred embodiment, users can directly access data (viaHypertext links for example) residing on Internet databases using a HTMLinterface provided by Web browsers and Web servers.

The computing and/or comparison module provides a computer readablecomparison result that can be processed in computer readable form bypredefined criteria, or criteria defined by a user, to provide contentbased in part on the comparison result that may be stored and output asrequested by a user using an output module, e.g., a display module.

In some embodiments, the content displayed on the display module can bethe relative levels of P2Y14 and/or UDP-glucose in the test sampleobtained from a subject as compared to a reference level. In certainembodiments, the content displayed on the display module can indicatewhether the levels of P2Y14 and/or UDP-glucose are found to bestatistically significantly higher in the test sample obtained from asubject as compared to a reference level. In some embodiments, thecontent displayed on the display module can show the levels of P2Y14and/or UDP-glucose from the subject measured at multiple time points,e.g., in the form of a graph. In some embodiments, the content displayedon the display module can indicate whether the subject has renalinflammation. In certain embodiments, the content displayed on thedisplay module can indicate whether the subject is in need of atreatment for renal inflammation.

In one embodiment, the content based on the computing and/or comparisonresult is displayed on a computer monitor. In one embodiment, thecontent based on the computing and/or comparison result is displayedthrough printable media. The display module can be any suitable deviceconfigured to receive from a computer and display computer readableinformation to a user. Non-limiting examples include, for example,general-purpose computers such as those based on Intel PENTIUM-typeprocessor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISCprocessors, any of a variety of processors available from Advanced MicroDevices (AMD) of Sunnyvale, Calif., or any other type of processor,visual display devices such as flat panel displays, cathode ray tubesand the like, as well as computer printers of various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the computing/comparisonresult. It should be understood that other modules can be adapted tohave a web browser interface. Through the Web browser, a user canconstruct requests for retrieving data from the computing/comparisonmodule. Thus, the user will typically point and click to user interfaceelements such as buttons, pull down menus, scroll bars and the likeconventionally employed in graphical user interfaces.

Systems and computer readable media described herein are merelyillustrative embodiments of the technology relating to determining thelevels of P2Y14 and/or UDP-glucose, and therefore are not intended tolimit the scope of the invention. Variations of the systems and computerreadable media described herein are possible and are intended to fallwithin the scope of the invention.

The modules of the machine, or those used in the computer readablemedium, may assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

Devices/Kits

Provided herein are kits and devices for practicing the assays andmethods described herein.

In some embodiments, described herein is a device for measuring theP2Y14 level in a test sample from a subject, the device comprising: (a)a P2Y14-specific antibody or antigen-binding portion thereof; and (b) atleast one solid support, wherein the antibody or antigen-binding portionthereof of part (a) are deposited on the support. In some embodiments,the device can perform an assay in which an antibody-protein orantibody-peptide complex is formed. In some embodiments, the solidsupport can be in the format of a dipstick, a microfluidic chip, amulti-well plate or a cartridge. The kits or devices can employimmuno-based lateral flow technology to produce a signal.

In some embodiments, described herein is a kit comprising: a device asdescribed in the preceding paragraph; and at least a detection antibody.In some embodiments, the detection antibody can be specific for P2Y14.In some embodiments, the detection antibody can be detectably labeled.In some embodiments, the kit can further comprise at least an agent forproducing a detectable signal from the detection antibody.

In some embodiments, the kit or device can comprise a reference, e.g. areference sample or reference signal. In some embodiments, the referencesample can comprise a urine sample from a healthy subject.

Methods of Treatment

In some embodiments, when the subject is identified as having renalinflammation, the assay or method further comprises administering to thesubject a treatment appropriate for treating renal inflammation. Renalinflammation, also called nephritis, can include several types such asglomerulonephritis, membranoproliferative glomerulonephritis,interstitial nephritis, IgA nephropathy, pyelonephritis, autoimmunedisorders related to CKD and inflammation, lupus nephritis,Goodpasture's syndrome, and Wegener's granulomatosis.

P2Y14 can be targeted for the treatment of renal inflammation.Accordingly, in some embodiments, the treatment appropriate for treatingrenal inflammation comprises a P2Y14 inhibitor.

In some embodiments, the P2Y14 inhibitor is a small or large organic orinorganic molecule. As used herein, the term “small molecule” refers tonatural or synthetic molecules having a molecular mass of less thanabout 5 kD, organic or inorganic compounds having a molecular mass ofless than about 5 kD, less than about 2 kD, or less than about 1 kD. Inone embodiment, the P2Y14 inhibitor is a 4,7-disubstituted naphthoicacid derivative. In one embodiment, the P2Y14 inhibitor is PPTN, i.e.,4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoicacid. Details about PPTN as a P2Y14 inhibitor can be found in Gauthier JY, et al., Bioorg Med Chem Lett., 2011, 21:2836-2839, the contents ofwhich are incorporated herein by reference in their entirety. In oneembodiment, the P2Y14 inhibitor is Prodrug 7j hydrochloride, which canbe available, e.g., from Axon Medchem.

In some embodiments, the P2Y14 inhibitor can be an anti-P2Y14 antibodymolecule or an antigen-binding fragment thereof. Suitable antibodiesinclude, but are not limited to, polyclonal, monoclonal, chimeric,humanized, recombinant, single chain, Fab, Fab′, F_(sc), R_(v), andF_((ab′)2) fragments. In some embodiments, neutralizing antibodies canbe used as inhibitors of P2Y14. Antibodies are readily raised in animalssuch as rabbits or mice by immunization with the antigen. Immunized miceare particularly useful for providing sources of B cells for themanufacture of hybridomas, which in turn are cultured to produce largequantities of monoclonal antibodies. In general, an antibody moleculeobtained from humans can be classified in one of the immunoglobulinclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all such classes,subclasses and types of human antibody species.

Antibodies provide high binding avidity and unique specificity to a widerange of target antigens and haptens. Monoclonal antibodies useful inthe practice of the methods disclosed herein include whole antibody andfragments thereof and are generated in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis.

The extracellular domain of the P2Y14 polypeptide, or a portion orfragment thereof, can serve as an antigen, and additionally can be usedas an immunogen to generate antibodies that immunospecifically bind theantigen, using standard techniques for polyclonal and monoclonalantibody preparation. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, or at least 15 amino acid residues, or atleast 20 amino acid residues, or at least 30 amino acid residues.

Useful monoclonal antibodies and fragments can be derived from anyspecies (including humans) or can be formed as chimeric proteins whichemploy sequences from more than one species. Human monoclonal antibodiesor “humanized” murine antibody can also be used in accordance with thepresent invention. For example, murine monoclonal antibody can be“humanized” by genetically recombining the nucleotide sequence encodingthe murine Fv region (i.e., containing the antigen binding sites) or thecomplementarily determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region. Humanizedtargeting moieties are recognized to decrease the immunoreactivity ofthe antibody or polypeptide in the host recipient, permitting anincrease in the half-life and a reduction in the possibility of adverseimmune reactions in a manner similar to that disclosed in EuropeanPatent Application No. 0,411,893 A2. The murine monoclonal antibodiesshould preferably be employed in humanized form. Antigen bindingactivity is determined by the sequences and conformation of the aminoacids of the six complementarily determining regions (CDRs) that arelocated (three each) on the light and heavy chains of the variableportion (Fv) of the antibody. The 25-kDa single-chain Fv (scFv)molecule, composed of a variable region (VL) of the light chain and avariable region (VH) of the heavy chain joined via a short peptidespacer sequence, is the smallest antibody fragment developed to date.Techniques have been developed to display scFv molecules on the surfaceof filamentous phage that contain the gene for the scFv. scFv moleculeswith a broad range of antigenic-specificities can be present in a singlelarge pool of scFv-phage library.

Chimeric antibodies are immunoglobin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobin constant region is derived from a human immunoglobinmolecule. Preferably, both regions and the combination have lowimmunogenicity as routinely determined.

In some embodiments, the P2Y14 inhibitor is a nucleic acid or a nucleicacid analog or derivative thereof, also referred to as a nucleic acidagent herein. In the context of this disclosure, the term “nucleic acid”refers to a polymer or oligomer of nucleotide or nucleoside monomersconsisting of naturally occurring bases, sugars and intersugar linkages.The term “nucleic acid” or “oligonucleotide” or “polynucleotide” areused interchangeably herein and can mean at least two nucleotidescovalently linked together. As will be appreciated by those skilled inthe art, the depiction of a single strand also defines the sequence ofthe complementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand.

Without limitation, the nucleic acid agent can be single-stranded ordouble-stranded. A single-stranded nucleic acid agent can havedouble-stranded regions, e.g., where there is internalself-complementarity, and a double-stranded nucleic acid agent can havesingle-stranded regions. The nucleic acid can be of any desired length.In particular embodiments, nucleic acid can range from about 10 to 100nucleotides in length. In various related embodiments, nucleic acidagents, single-stranded, double-stranded, and triple-stranded, can rangein length from about 10 to about 50 nucleotides, from about 20 to about50 nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length. In some embodiments, nucleic acid agentis from about 9 to about 39 nucleotides in length. In some otherembodiments, nucleic acid agent is at least 30 nucleotides in length.

The nucleic acid agent can comprise modified nucleosides as known in theart. Modifications can alter, for example, the stability, solubility, orinteraction of the nucleic acid agent with cellular or extracellularcomponents that modify activity. In certain instances, it can bedesirable to modify one or both strands of a double-stranded nucleicacid agent. In some cases, the two strands will include differentmodifications. In other instances, multiple different modifications canbe included on each of the strands. The various modifications on a givenstrand can differ from each other, and can also differ from the variousmodifications on other strands. For example, one strand can have amodification, and a different strand can have a different modification.In other cases, one strand can have two or more different modifications,and the another strand can include a modification that differs from theat least two modifications on the first strand.

Single-stranded and double-stranded nucleic acid agents that areeffective in inducing RNA interference are referred to as siRNA, RNAiagents, iRNA agents, or RNAi inhibitors herein. As used herein, the term“iRNA agent” refers to a nucleic acid agent which can mediate thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway.

In some embodiments, the P2Y14 inhibitor is an antisenseoligonucleotide. One of skill in the art is well aware thatsingle-stranded oligonucleotides can hybridize to a complementary targetsequence and prevent access of the translation machinery to the targetRNA transcript, thereby preventing protein synthesis. Thesingle-stranded oligonucleotide can also hybridize to a complementaryRNA and the RNA target can be subsequently cleaved by an enzyme such asRNase H and thus preventing translation of target RNA. Alternatively, orin addition to, the single-stranded oligonucleotide can modulate theexpression of a target sequence via RISC mediated cleavage of the targetsequence, i.e., the single-stranded oligonucleotide acts as asingle-stranded RNAi agent. A “single-stranded RNAi agent” as usedherein, is an RNAi agent which is made up of a single molecule. Asingle-stranded RNAi agent can include a duplexed region, formed byintra-strand pairing, e.g., it can be, or include, a hairpin orpan-handle structure.

In general, any method of delivering a nucleic acid molecule can beadapted for use with the nucleic acid agents described herein.

In some embodiments, the P2Y14 inhibitor can also be a peptide, apeptidomimetic, a protein, a monosaccharide, a disaccharide, atrisaccharide, an oligosaccharide, a polysaccharide, a lipid, aglycosaminoglycan, an extract made from a biological material, or anycombinations thereof.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosages of P2Y14 inhibitors for use inhumans. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The term “effective amount” as used herein refers to the amount of atherapy needed to alleviate at least one or more symptoms of the diseaseor disorder (e.g., inflammation or renal inflammation), and relates to asufficient amount of pharmacological composition to provide the desiredeffect. The term “therapeutically effective amount” therefore refers toan amount of a therapy that is sufficient to cause a particular effectwhen administered to a typical subject. An effective amount as usedherein, in various contexts, would also include an amount sufficient todelay the development of a symptom of the disease, alter the course of asymptom of the disease (for example but not limited to, slowing theprogression of a symptom of the disease), or reverse a symptom of thedisease. Thus, it is not generally practicable to specify an exact“effective amount”. However, for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing only routine experimentation.

In some embodiments, an effective amount of a P2Y14 inhibitor can be anamount which causes the level of P2Y14 expression to decrease or, atleast, to increase at a lower rate than it would be expected to increasein a subject not receiving the P2Y14 inhibitor. In some embodiments, aneffective amount can be an amount that decreases the amount of P2Y14polypeptide present in the subject and/or P2Y14 polypeptide present in asample (e.g., urine) obtained from a subject by a statisticallysignificant amount.

In some embodiments, an effective amount of a P2Y14 inhibitor can be anamount which reduces the extent of renal inflammation.

In some embodiments, an effective amount of a P2Y14 inhibitor can be anamount that decreases the expression or level of pro-inflammatorychemokines in ICs.

The dosage can be determined by one of skill in the art and can also beadjusted by the individual physician in the event of any complication.Typically, the dosage of a composition comprising a P2Y14 inhibitordisclosed herein can range from 0.001 mg/kg body weight to 5 g/kg bodyweight. In some embodiments, the dosage range is from 0.001 mg/kg bodyweight to 1 g/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kgbody weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg bodyweight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kgbody weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg bodyweight to 0.1 mg/kg body weight, or from 0.001 mg/kg body weight to0.005 mg/kg body weight. Alternatively, in some embodiments the dosagerange is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kgbody weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kgbody weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5g/kg body weight, or from 4.5 g/kg body weight to 5 g/kg body weight.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test bioassays or systems. The dosageshould not be so large as to cause unacceptable adverse side effects.

In some embodiments, the P2Y14 inhibitors described herein can improverenal function. For example, renal function is improved by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 200%, orat least 300%. Measurable markers of renal function, are well known inthe medical and veterinary literature and to those of skill in the art,and include, but are not limited to, blood urea nitrogen or “BUN” levels(both static measurements and measurements of rates of increase ordecrease in BUN levels), serum creatinine levels (both staticmeasurements and measurements of rates of increase or decrease in serumcreatinine levels), measurements of the BUN/creatinine ratio (staticmeasurements of measurements of the rate of change of the BUN/creatinineratio), urine/plasma ratios for creatinine, urine/plasma ratios forurea, glomerular filtration rates (GFR), serum concentrations of sodium(Na⁺), urine osmolarity, daily urine output, and the like. Of the above,measurements of the plasma concentrations of creatinine and/or urea orBUN are particularly important and useful readouts of renal function.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., disclosed hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

Although any known methods, devices, and materials may be used in thepractice or testing of the invention, the methods, devices, andmaterials in this regard are disclosed herein.

Some embodiments of the invention are listed in the following numberedparagraphs:

paragraph 1. An assay comprising:(i) measuring, in a sample obtained from a subject, a level of P2Y14;(ii) comparing the level of P2Y14 with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of P2Y14 is above the reference level; and (b) not having renalinflammation if the level of P2Y14 is at or below the reference level.paragraph 2. The assay of paragraph 1, wherein when the level of P2Y14is above the reference level, the method further comprises providing atreatment appropriate for treating renal inflammation.paragraph 3. The assay of paragraph 2, wherein the treatment comprises aP2Y14 inhibitor.paragraph 4. The assay of any one of the preceding paragraphs, whereinthe level of P2Y14 is a protein level.paragraph 5. The assay of paragraph 4, wherein the level of P2Y14 ismeasured by an immunoassay.paragraph 6. The assay of paragraph 5, wherein the sample is contactedwith an anti-P2Y14 antibody.paragraph 7. The assay of paragraph 6, wherein the anti-P2Y14 antibodyis detectably labeled or capable of generating a detectable signal.paragraph 8. The assay of paragraph 6 or 7, wherein the antibody isfluorescently labeled.paragraph 9. The assay of any one of paragraphs 1-3, wherein the levelof P2Y14 is measured by measuring a nucleic acid encoding P2Y14.paragraph 10. The assay of any one of the preceding paragraphs, whereinthe reference level is an average P2Y14 level in a population of healthysubjects.paragraph 11. The assay of any one of paragraphs 1-9, wherein thereference level is two standard deviations above an average P2Y14 levelin a population of healthy subjects.paragraph 12. The assay of any one of the preceding paragraphs, whereinthe assay detects early stage inflammation.paragraph 13. The assay of any one of the preceding paragraphs, furthercomprising measuring a level of UDP-glucose in a sample obtained fromthe subject, and comparing the level of UDP-glucose with a referencelevel.paragraph 14. The assay of any one of the preceding paragraphs, whereinthe sample is a urine sample.paragraph 15. The assay of any one of the preceding paragraphs, whereinthe subject is a mammal.paragraph 16. The assay of paragraph 15, wherein the mammal is a human.paragraph 17. A method of detecting renal inflammation of a subject, themethod comprising(i) assaying, in a sample obtained from a subject, a level of P2Y14;(ii) comparing the level of P2Y14 with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of P2Y14 is above the reference level; and (b) not having renalinflammation if the level of P2Y14 is at or below the reference level.paragraph 18. The method of paragraph 17, wherein when the level ofP2Y14 is above the reference level, the method further comprisesproviding a treatment appropriate for treating renal inflammation.paragraph 19. The method of paragraph 18, wherein the treatmentcomprises a P2Y14 inhibitor.paragraph 20. The method of any one of paragraphs 17-19, wherein thelevel of P2Y14 is a protein level.paragraph 21. The method of paragraph 20, wherein the level of P2Y14 ismeasured by an immunoassay.paragraph 22. The method of paragraph 21, wherein the sample iscontacted with an anti-P2Y14 antibody.paragraph 23. The method of paragraph 22, wherein the anti-P2Y14antibody is detectably labeled or capable of generating a detectablesignal.paragraph 24. The method of paragraph 22 or 23, wherein the antibody isfluorescently labeled.paragraph 25. The method of any one of paragraphs 17-19, wherein thelevel of P2Y14 is measured by measuring a nucleic acid encoding P2Y14.paragraph 26. The method of any one of paragraphs 17-25, wherein thereference level is an average P2Y14 level in a population of healthysubjects.paragraph 27. The method of any one of paragraphs 17-25, wherein thereference level is two standard deviations above an average P2Y14 levelin a population of healthy subjects.paragraph 28. The method of any one of paragraphs 17-27, wherein themethod detects early stage inflammation.paragraph 29. The method of any one of paragraphs 17-28, furthercomprising measuring a level of UDP-glucose in a sample obtained fromthe subject, and comparing the level of UDP-glucose with a referencelevel.paragraph 30. The method of any one of paragraphs 17-29, wherein thesample is a urine sample.paragraph 31. The method of any one of paragraphs 17-30, wherein thesubject is a mammal.paragraph 32. The method of paragraph 31, wherein the mammal is a human.paragraph 33. A method of monitoring treatment progress in a subjectsuffering from renal inflammation, the method comprising:(i) measuring, at a first time point, a first level of P2Y14 in a firstsample obtained from the subject;(ii) administering to the subject a therapeutic agent for treating renalinflammation; and(iii) measuring, at a second time point, a second level of P2Y14 in asecond sample obtained from the subject, wherein the second time pointis later than the first time point and after said administration, andwherein if the second level is significantly lower than the first level,then the treatment is considered to be effective.paragraph 34. The method of paragraph 33, wherein the first sample andthe second sample are urine samples.paragraph 35. The method of paragraph 33 or 34, wherein the therapeuticagent is a P2Y14 inhibitor.paragraph 36. The method of any one of paragraphs 33-35, wherein thesubject is a human.paragraph 37. A method of treating a subject having renal inflammation,the method comprising administering a P2Y14 inhibitor to the subject.paragraph 38. The method of paragraph 37, wherein the P2Y14 inhibitor isPPTN or an anti-P2Y14 antibody.paragraph 39. The method of paragraph 37 or 38, wherein the subject is ahuman.paragraph 40. A method of treating a subject determined to have a levelof P2Y14 above a reference level, the method comprising administering atreatment appropriate for treating renal inflammation.paragraph 41. The method of paragraph 40, wherein the treatmentappropriate for treating renal inflammation comprises a P2Y14 inhibitor.paragraph 42. The method of paragraph 41, wherein the P2Y14 inhibitor isPPTN or an anti-P2Y14 antibody.paragraph 43. The method of any one of paragraphs 40-42, wherein thesubject is a human.paragraph 44. An assay comprising:(i) measuring, in a sample obtained from a subject, a level ofUDP-glucose;(ii) comparing the level of UDP-glucose with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of UDP-glucose is above the reference level; and (b) not havingrenal inflammation if the level of UDP-glucose is at or below thereference level.paragraph 45. The assay of paragraph 44, wherein when the level ofUDP-glucose is above the reference level, the method further comprisesproviding a treatment appropriate for treating renal inflammation.paragraph 46. The assay of paragraph 45, wherein the treatment comprisesa P2Y14 inhibitor.paragraph 47. The assay of any one of paragraphs 44-46, wherein thereference level is an average UDP-glucose level in a population ofhealthy subjects.paragraph 48. The assay of any one of paragraphs 44-46, wherein thereference level is two standard deviations above an average UDP-glucoselevel in a population of healthy subjects.paragraph 49. The assay of any one of paragraphs 44-48, wherein theassay detects early stage inflammation.paragraph 50. The assay of any one of paragraphs 44-49, wherein thesample is a urine sample.paragraph 51. The assay of any one of paragraphs 44-50, wherein thesubject is a mammal.paragraph 52. The assay of paragraph 51, wherein the mammal is a human.paragraph 53. A method of detecting renal inflammation of a subject, themethod comprising(i) assaying, in a sample obtained from a subject, a level ofUDP-glucose;(ii) comparing the level of UDP-glucose with a reference level; and(iii) identifying the subject as (a) having renal inflammation if thelevel of UDP-glucose is above the reference level; and (b) not havingrenal inflammation if the level of UDP-glucose is at or below thereference level.paragraph 54. The method of paragraph 53, wherein when the level ofUDP-glucose is above the reference level, the method further comprisesproviding a treatment appropriate for treating renal inflammation.paragraph 55. The method of paragraph 54, wherein the treatmentcomprises a P2Y14 inhibitor.paragraph 56. The method of any one of paragraphs 53-55, wherein thereference level is an average UDP-glucose level in a population ofhealthy subjects.paragraph 57. The method of any one of paragraphs 53-55, wherein thereference level is two standard deviations above an average UDP-glucoselevel in a population of healthy subjects.paragraph 58. The method of any one of paragraphs 53-57, wherein themethod detects early stage inflammation.paragraph 59. The method of any one of paragraphs 53-58, wherein thesample is a urine sample.paragraph 60. The method of any one of paragraphs 53-59, wherein thesubject is a mammal.paragraph 61. The method of paragraph 60, wherein the mammal is a human.paragraph 62. A method of monitoring treatment progress in a subjectsuffering from renal inflammation, the method comprising:(i) measuring, at a first time point, a first level of UDP-glucose in afirst sample obtained from the subject;(ii) administering to the subject a therapeutic agent for treating renalinflammation; and(iii) measuring, at a second time point, a second level of UDP-glucosein a second sample obtained from the subject, wherein the second timepoint is later than the first time point and after said administration,and wherein if the second level is significantly lower than the firstlevel, then the treatment is considered to be effective.paragraph 63. The method of paragraph 62, wherein the first sample andthe second sample are urine samples.paragraph 64. The method of paragraph 62 or 63, wherein the therapeuticagent is a P2Y14 inhibitor.paragraph 65. The method of any one of paragraphs 62-64, wherein thesubject is a human.paragraph 66. A method of treating a subject determined to have a levelof UDP-glucose above a reference level, the method comprisingadministering a treatment appropriate for treating renal inflammation.paragraph 67. The method of paragraph 66, wherein the treatmentappropriate for treating renal inflammation comprises a P2Y14 inhibitor.paragraph 68. The method of paragraph 67, wherein the P2Y14 inhibitor isPPTN or an anti-P2Y14 antibody.paragraph 69. The method of any one of paragraphs 66-68, wherein thesubject is a human.

Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areuseful to an embodiment, yet open to the inclusion of unspecifiedelements, whether useful or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

The terms “disease”, “disorder”, or “condition” are used interchangeablyherein, refer to any alternation in state of the body or of some of theorgans, interrupting or disturbing the performance of the functionsand/or causing symptoms such as discomfort, dysfunction, distress, oreven death to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, affectation.

As used herein, the term “renal inflammation” extends to all conditionswhich are substantially characterized by the occurrence of inflammationwithin the kidney, or where the occurrence of inflammation in the kidneyis caused by a disease or an inflammatory condition which primarilyaffects a site in the body other than the kidney. In particular,inflammation may occur at a site including, but not limited to; theglomerulus, Bowman's capsule or Bowman's space. Typically, theinflammation results in at least partial impairment of kidney functionand/or kidney failure.

Examples of specific conditions which fall within the meaning of theterm “renal inflammation” include, but are not limited to: renaldisorders which include, but are not limited to: chronic renal failure,acute renal failure, heterologous nephrotoxic nephritis,glomerulonephritis, sclerosis of the glomerulus, systemic lupuserythematosus (SLE), diabetic nephropathy, diabetic nephropathy whereinthe diabetic nephropathy accompanies sclerosis of the liver, andglomerulonephritis wherein the glomerulonephritis is accompanied bysclerosis of the liver.

In some embodiments, renal inflammation can relate to an immune-mediateddisease which affects the cells of the kidney and/or kidney function.Such conditions can include, but are not limited to: Immunoglobulin Anephropathy, membranoproliferative glomerulonephritis, mesangialproliferative glomerulonephritis, nonproliferative glomerulonephritis,membranous glomerulonephritis, minimal-change disease, primary focalsegmental glomerulosclerosis (FSGS), fibrillary glomerulonephritis,immunotactoid glomerulonephritis, proliferative glomerulonephritis,progressive glomerulonephritis, anti-GBM disease, kidney ischemia,kidney vasculitis, including disease associated with anti-neutrophilcytoplasmic antibodies (ANCA) (e.g., Wegener granulomatosis), lupusnephritis cryoglobulinemia-associated glomerulonephritis, bacterialendocarditis, Henoch-Schönlein purpura, postinfectiousglomerulonephritis, Hepatitis C, diabetic nephropathy, myloidosis,hypertensive nephrosclerosis, light-chain disease from multiple myeloma,secondary focal glomerulosclerosis, and hypertensive nephrosclerosis.

As used herein, the term “early stage” with respect to inflammationmeans that the tissue or organ exhibits minimal or mild damage resultingfrom inflammation. “Early stage inflammation” can mean a clinical stageof inflammation where substantial infiltration of pro-inflammatory cellsand/or molecules into renal tissues has not occurred.

As used herein, the term “antibody” refers to an immunoglobulin moleculeand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybind an antigen. The terms also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms besides antibodies; including, for example, Fv,Fab, and F(ab)′2 as well as bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883(1988) and Bird et al., Science 242, 423-426 (1988), which areincorporated herein by reference). (See, generally, Hood et al.,Immunology, Benjamin, N.Y., 2ND ed. (1984), Harlow and Lane, Antibodies.A Laboratory Manual, Cold Spring Harbor Laboratory (1988) andHunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporatedherein by reference). In some embodiments, antibody reagents, e.g.antibodies, monoclonal and chimeric antibodies useful in the methods asdisclosed herein can be manufactured using well-known methods, e. g., asdescribed in Howard and Kaser “Making and Using Antibodies: A PracticalHandbook” CRC Press (2006); which is incorporated by reference herein inits entirety.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein to refer to one or more fragments of afull length antibody that retain the ability to specifically bind to atarget of interest. Examples of binding fragments encompassed within theterm “antigen-binding fragment” of a full length antibody include (i) aFab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fabfragments linked by a disulfide bridge at the hinge region; (iii) an Fdfragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546; which isincorporated by reference herein in its entirety), which consists of aVH or VL domain; and (vi) an isolated complementarity determining region(CDR) that retains specific antigen-binding functionality. Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VH regions pair to form monovalent molecules knownas single chain Fv (scFv). See e.g., U.S. Pat. Nos. 5,260,203,4,946,778, and 4,881,175; Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibodyfragments can be obtained using any appropriate technique includingconventional techniques known to those of skill in the art. The term“monospecific antibody” refers to an antibody that displays a singlebinding specificity and affinity for a particular target, e.g., epitope.This term includes a “monoclonal antibody” or “monoclonal antibodycomposition,” which as used herein refer to a preparation of antibodiesor fragments thereof of single molecular composition, irrespective ofhow the antibody was generated.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, “specific binding” can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnon-target entity. In some embodiments, “specific binding” refers to anantibody binding with a dissociation constant (K_(D)) of 10⁻⁵M or less,10⁻⁶M or less, or 10⁻⁷M or less, and binding to the predeterminedantigen with a K_(D) that is at least twofold less than its K_(D) forbinding to a nonspecific antigen other than the predetermined antigen.

As used herein, the terms “proteins” and “polypeptides” are usedinterchangeably to designate a series of amino acid residues connectedto each other by peptide bonds between the alpha-amino and carboxygroups of adjacent residues. The terms “protein”, and “polypeptide”refer to a polymer of amino acids, including modified amino acids (e.g.,phosphorylated, glycated, glycosylated, etc.) and amino acid analogs,regardless of its size or function. “Protein” and “polypeptide” areoften used in reference to relatively large polypeptides, whereas theterm “peptide” is often used in reference to small polypeptides, butusage of these terms in the art overlaps. The terms “protein” and“polypeptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary polypeptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

As used herein, the terms “inhibitor of P2Y14” or “P2Y14 inhibitor”refer to an agent that can decrease the expression level and/or activityof P2Y14, e.g. by at least 5%, at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99% or more. It is preferred that aninhibitor of P2Y14 inhibits P2Y14 activity without substantiallyinhibiting other receptors or activities. It is also preferred that thespecific inhibitory activity occurs at a concentration that is not toxicto the subject. In some embodiments, a P2Y14 inhibitor can decrease thelevel of P2Y14 mRNA, the level of P2Y14 polypeptide, and/or the level ofsignaling of P2Y14 in the cell of origin or a second cell. P2Y14activity can be monitored, e.g., by measuring calcium influx and/or bymeasuring ligand binding. In some embodiments, a P2Y14 inhibitor canspecifically bind a P2Y14 polypeptide. In some embodiments, a P2Y14inhibitor can reduce signal transduction mediated by P2Y14. Irreversibleor reversible inhibitors of P2Y14 can be used in the methods disclosedherein.

As used herein, the term “anti-P2Y14 antibody” means an antibody, e.g.,an IgG molecule, that binds specifically to the extracellular portion ofa full length P2Y14 polypeptide.

The terms “decrease”, “reduce”, “reduction”, or “inhibit” are all usedherein generally to mean a decrease by a statistically significantamount. However, for the avoidance of doubt, “decrease”, “reduce”,“reduction”, or “inhibit” can mean a decrease by at least 10% ascompared to a reference level, for example a decrease by at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% or up to and including a 100% decrease (e.g.absent level or non-detectable level as compared to a reference level),or any decrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom is meant a decrease by a statisticallysignificant amount in such level. The decrease can be, for example, atleast 10%, at least 20%, at least 30%, at least 40% or more, and ispreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, or “enhance” are all used herein togenerally mean an increase by a statically significant amount; for theavoidance of doubt, the terms “increased”, “increase”, or “enhance”, canmean an increase of at least 10% as compared to a reference level, forexample an increase of at least about 10%, at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. In the context of a marker or symptom is meant astatistically significant increase in such level.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of renal inflammation. The term “treating”includes reducing or alleviating at least one adverse effect or symptomof renal inflammation. Treatment is generally “effective” if one or moresymptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted. Thatis, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation of, or at least slowing of, progress orworsening of symptoms compared to what would be expected in the absenceof treatment. Beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, remission (whether partial or total), and/ordecreased mortality, whether detectable or undetectable. For example,treatment is considered effective if the extent or amount of renalinflammation is reduced, or the progression of renal inflammation ishalted. In another example, treatment is considered effective if renalfunction is improved. The term “treatment” of a disease also includesproviding relief from the symptoms or side-effects of the disease(including palliative treatment).

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, and canine species, e.g., dog, fox, wolf. In someembodiments, the subject is a mammal, e.g., a primate, e.g., a human.The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) difference.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject, e.g. parenteral, intravenous,intralesional, or intratumoral.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection. The administration can be systemic or local.Therapeutic agents can be placed in a tissue or organ-specific deliverydevice. For example, a P2Y14 inhibitor can be placed in a renal catheterfor direct delivery or to the renal circulation.

The term “software” is used interchangeably herein with “program” andrefers to prescribed rules to operate a computer. Examples of softwareinclude: software; code segments; instructions; computer programs; andprogrammed logic.

The term a “computer system” may refer to a system having a computer,where the computer comprises a computer-readable medium embodyingsoftware to operate the computer.

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);Immunology by Werner Luttmann, published by Elsevier, 2006. Definitionsof common terms in molecular biology can also be found in BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); Current Protocols in Protein Science (CPPS) (JohnE. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocolsin Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley andSons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique byR. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal CellCulture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather andDavid Barnes editors, Academic Press, 1st edition, 1998) which are allincorporated by reference herein in their entireties.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1% of the value being referred to. For example, about 100 meansfrom 99 to 101.

Although methods and materials similar or equivalent to those disclosedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The abbreviation,“e.g.” is derived from the Latin exempli gratia, and is used herein toindicate a non-limiting example. Thus, the abbreviation “e.g.” issynonymous with the term “for example.”

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydisclosed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredisclosed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments disclosed herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The technologydisclosed herein is further illustrated by the following examples whichin no way should be construed as being further limiting.

Example 1: Renal Intercalated Cells Sense and Mediate SterileInflammation Via the P2Y14 Receptor

Uncontrolled inflammation is one of the leading causes of kidneyfailure. Pro-inflammatory responses can occur in the absence ofinfection, a process called sterile inflammation. It is shown hereinthat the purinergic receptor P2Y14 (GPR105) is specifically and highlyexpressed in collecting duct intercalated cells (ICs), and mediatessterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, adamage-associated molecular pattern molecule (DAMP) released by injuredcells. Using transgenic mice expressing EGFP in ICs, as well as culturedcells, it was found that UDP-glucose activates the MEK1/2-ERK1/2pathway, and increases pro-inflammatory chemokine expression. Theseeffects were prevented following inhibition of P2Y14 with the smallmolecule PPTN. Tail vein injection of mice with UDP-glucose induced therecruitment of neutrophils to the renal medulla. This study identifiesICs as novel sensors, mediators and effectors of inflammation in thekidney via P2Y14.

In this study elevated expression of a restricted number of P2 receptorsin ICs was uncovered, most notably P2Y14. Moreover, P2Y14 expression wasnot detectable in other renal epithelial cells. Evidence was alsoprovided that P2Y14 activation by UDP-glucose induces a pro-inflammatoryresponse in ICs that is mediated by activation of the MAPK pathway. Thisis followed by increased expression of pro-inflammatory chemokines inICs, and subsequent neutrophil infiltration in the renal medulla. Thus,a novel inflammatory role for renal ICs via P2Y14 signaling has beenidentified.

Methods Reagents and Antibodies

Uridine 5′-diphosphoglucose disodium salt hydrate from Saccharomycescerevisiae (UDP-glucose) and the MEK inhibitor PD98059 were purchasedfrom Sigma Aldrich (St. Louis, Mo.). Uridine diphospho-D-[6-³H] glucose([³H]UDP-glucose) was purchased from Perkin Elmer (Waltham, Mass.).PPTN, a selective high affinity antagonist of the P2Y14 receptor hasbeen described previously (50). The chicken antibody against theV-ATPase B1 subunit has been described previously (72). Anaffinity-purified chicken antibody against the V-ATPase A subunit wasraised against the same sequence as previously described for a rabbitanti-V-ATPase A subunit antibody and was found to have the samespecificity (73, 74). A rabbit anti-P2Y14 antibody and its immunizingpeptide were purchased from Alomone labs (Jerusalem, Israel). Rabbitanti-cytoskeletal actin antibody was from Bethyl Laboratory (Montgomery,Tex.). Rabbit anti-pendrin antibody was a kind gift from Dr. Aronson(Yale University). Rabbit monoclonal antibodies against p-p44/p42 MAPKand against total p44/p42 MAPK were purchased from Cell SignalingTechnology (Boston, Mass.). Rat anti-Ly6G antibody was from Biolegend(San Diego, Calif.). Mouse anti-pan-actin was purchased from EMDMillipore (Billerica, Mass.). Affinity purified anti-rabbit HRP,anti-chicken HRP, anti-mouse HRP, anti-rabbit cy3 and anti-chicken cy3were all raised in donkeys and purchased from Jackson Immunoresearch(West Grove, Pa.). Affinity purified anti-mouse HRP and anti-rabbit FITCwere raised in goats and purchased from Jackson Immunoresearch. A goatanti-rat cy3 was from Invitrogen (Grand Island, N.Y.).Streptavidin-fluorescein was purchased from Invitrogen. Cell culturemedium was purchased from Invitrogen, and bovine serum was purchasedfrom Atlanta Biologicals (Lawrenceville, Calif.).

Animals

Adult male mice (eight to ten weeks old) were used for all experiments.Transgenic mice expressing EGFP under the promoter of the V-ATPase B1subunit (B1-EGFP) mice have been described previously (35). Wild type(C57BL/6×CBAF1) mice were purchased from Jackson Laboratory (Bar Harbor,Me.). Animals were housed under standard conditions and maintained on astandard rodent diet. The Massachusetts General Hospital (MGH)Subcommittee on Research Animal Care approved all animal studies, inaccordance with National Institutes of Health, Department ofAgriculture, and Accreditation of Laboratory Animal Care requirements.For tail vein injections, animals were kept under isofluorane anesthesia(Baxter, Deerfield, Ill.) for several minutes to allow the injection of200 μl of either saline solution or a saline solution containing 100 μMUDP-glucose (200 mg/kg of body weight).

Isolation of Intercalated Cells from Mouse Kidneys

Mice were anesthetized using pentobarbital sodium (50 mg/kg body, ip,Nembutal, Abbott Laboratories, Abbott Park, Ill.). The blood was flushedout of the organs by perfusing the animals with a phosphate-bufferedsaline (PBS) through the cardiac left ventricle at a constant flow rateof 17 ml/min. Kidneys were excised and sliced, and some kidneys weremicrodissected to separate the cortex from the medulla. Tissues werethen minced immediately with scissors in RPMI 1640 medium (Invitrogen,Grand Island, N.Y.) containing 1.0 mg/ml collagenase type I(Invitrogen), 1.0 mg/ml collagenase type II (Sigma Aldrich) and 2 mg/mlhyaluronidase (Sigma Aldrich), and digested for 45 min at 37° C. A40-μm-nylon mesh was used to remove undigested material following tissuedigestion. Cells were then washed once with RPMI 1640 medium and oncewith a calcium-free PBS. EGFP-positive (EGFP(+)) and negative (EGFP(−))cells were isolated immediately by FACS, based on their greenfluorescence intensity, as described previously (36). Cell isolation wasperformed at the MGH flow cytometry core facility using a modified FACSVantage cell sorter (BD Biosciences, San Jose, Calif.). FACS isolatedsamples were collected in PBS and used without delay for subsequenttreatment and/or cell fractionation, protein and RNA isolation, orimaging.

RNA Isolation and RT-PCR

Total RNA was isolated from cells using RNeasy micro kit (Qiagen,Valencia, Calif.) and from tissues using RNeasy Mini kit, as describedpreviously (36). DNase I digestion was performed using RNase-Free DNaseSet (Qiagen) according to the manufacturer's instructions. All reversetranscription (RT) and PCR reagents were from Applied Biosystems (FosterCity, Calif.). Reverse-transcription was performed for 1 h at 42° C. ina final volume of 50 μl with 1× buffer II, 5 mM MgCl₂, 1.0 mM each dNTP,1 U/μl RNase inhibitor, 2.5 μM random hexamers, and 2.5 U/μl MuLVreverse transcriptase. PCR was performed on RT products. The sequencesof the PCR primer sets, purchased from Invitrogen, are listed inTable 1. For end point PCRs, reaction mixtures consisted of a 20 μlfinal volume containing 2 μl template, 1.25 units AmpliTaq Gold DNApolymerase, 1× buffer II, 1.5 mM MgCl₂, 1.0 mM each dNTP, and 0.5 μMforward and reverse oligonucleotide primers. The following parameterswere used for PCR: 8 min at 95° C. to activate the polymerase, 35 cyclesof melting for 30 s at 95° C., annealing for 30 s at 60° C., extensionfor 30 s at 72° C., and a final extension for 10 min at 72° C. Theamplification products were visualized by electrophoresis on a 1-2%agarose gel containing GelStar stain (Lonza, Rockland, Me.).

Real Time PCR was performed with a 7300 Real Time PCR system (AppliedBiosystems). Amplification products were detected using the Power SYBRGreen PCR master mix (Applied Biosystems), according to themanufacturer's instructions. Standard-curve relative quantificationswere performed and relative values of each sample were normalized toGAPDH values. Samples were analyzed in triplicates for each experiment.

Flow Cytometry Analysis

Tissues and cells were prepared as described above for FACS. Prior toflow cytometry, cell suspensions were stained in PBS with BSA 1% usingthe following antibodies purchased from BD Biosciences (San Jose,Calif.): PE-conjugated anti-CD90 (clone 53-2.1), PE-conjugated anti-B220(clone RA3-6B2), PE-conjugated anti-CD49b (clone DX5), PE-conjugatedanti-NK1.1 (clone PK136), PE-conjugated anti-Ly-6G (clone 1A8),APC-Cy7-conjugated anti-CD11b (clone M1/70), PE-Cy7-conjugatedanti-F4/80 (clone BM8), Alexa Fluor 700-conjugated anti-CD11c (cloneHL3). Antibodies purchased from BD Pharmigen were also used:PE-conjugated anti-CD19 (clone 1D3), PE-Cy7-conjugated anti-B220 (cloneRA3-6B2), FITC-conjugated anti-CD3e (clone 145-2C11), PE-Cy7-conjugatedanti-CD4 (clone RM4-5) and PerCP-conjugated anti-CD8a (clone 53-6.7).Single cell suspensions were labeled for 45 min at 4° C. Formonocyte/neutrophil staining, the following PE-conjugated antibodieswere used: anti-CD90, anti-B220, anti-CD19, anti-CD49b, anti-NK1.1 andanti-Ly-6G. Neutrophils were defined as Lin+CD11b+ cells. B cells weredefined as B220+CD19+ cells. Total T cells were defined as CD3e+ cells.CD4 T cells were defined as CD3e+CD4+ cells. CD8 T cells were defined asCD3e+CD8a+ cells. The number of neutrophils, B and T cells was definedas the total number of cells per organ multiplied by the percentage ofeach cell type identified by flow cytometry (LSRII; BD Biosciences).Cell suspensions obtained from the spleen were labeled with appropriateantibodies for staining controls. Data were analyzed with FlowJo v.8.8.7(Tree Star, Inc., Ashland, Oreg.).

Cell Culture and Protein Preparation

MDCK-C11 cells were cultured at 37° C. in a 5% CO2-95% O2 mix in DMEM(Invitrogen) supplemented with 2 mM glutamine, 10% fetal bovine serum(Invitrogen), penicillin (100 U/ml), and streptomycin (100 μg/ml)(Invitrogen). Prior to any treatment, cells were serum starved for 24hours. For cell surface biotinylation assays, cells were either grown toconfluence on plastic dishes or on filters. Biotinylation was performedas described previously (75). Briefly cells were incubated with 1 mg/mlof biotin (Thermo Scientific, Pittsburgh, Pa.) in PBS pH 8 on ice for 1h. The excess biotin was quenched with 100 mM glycine and cells wereeither further processed for immunostaining or lysed in lysis buffer[150 mM NaCl, 5 mM EDTA, 50 mM Tris/HEPES, pH 7.5; 1% (vol/vol) TritonX-100] containing protease and phosphatase inhibitors (Complete MiniEDTA free, PhosSTOP, Roche Diagnostics, Laval, Canada), for 20 min at 4°C. Total protein expression was determined in 2-5 μg of proteins fromthe total cell extracts. 50-100 μg of cell lysate proteins wereincubated with neutravidin beads (Thermo Scientific) overnight at 4° C.(according to the manufacturer's protocol). After centrifugation (2,500g for 5 min) and removal of the supernatant, beads were washed threetimes with the lysis buffer, twice with a high-salt buffer (500 mM NaCl,5 mM EDTA, 50 mM Tris, 0.1% Triton X-100, pH 7.5) and once with ano-salt buffer (10 mM Tris, pH 7.5). Proteins were subjected to SDS-PAGEfollowing denaturation in Laemmli buffer for 5 min at 95° C. Forenzymatic deglycosylation, 20 μg of total and 100 μg of biotinylated andavidin precipitated proteins were treated for 1 h at 37° C. with eitherendoglycosydase H or PNGase F or control according to the manufacturer'sprotocol (New England Biolabs, Ipsxich, Mass.). For P2Y₁₄ antagoniststudies, PPTN was dissolved in DMSO and applied to confluent MDCK-C11cells at a final concentration of 10 μM (0.05% DMSO). Pretreatment withPPTN or vehicle (0.05% DMSO) was for 30 minutes prior to control orUDP-glucose treatment.

Radioligand Binding Assays

[³H]UDP-glucose binding assays were performed in MDCK-C11 cell line andFACS isolated IC membrane preparations, as previously described (16).Confluent MDCK-C11 cells were scrapped in ice-cold PBS, pelleted bycentrifugation (500 g, 10 min) and then resuspended in 1 ml ice-coldTris-acetate 0.2 M buffer (pH 7.5) containing protease inhibitors(Complete Mini, Roche, Indianapolis, Ind.) using a 25G needle. Cellmembranes were harvested by passing through a cell cracker (HGM labequipment, Heidelberg, Germany) 10 times. The solution was thencentrifuged 10 min at 17000 g and membrane pellets were frozen in liquidnitrogen and kept at −80° C. until use. Protein concentration wasdetermined using a nanodrop 2000 (Thermo scientific).

For dose-displacement assays 15 μg of MDCK-C11 cell membrane proteinswere incubated for 3 hours at 22° C. in a medium containing 50 mMTris/HCl pH 7.4, 1 mM EDTA, 5 mM MgCl2 and BSA (5 mg/ml),[³H]-UDP-glucose (3 nM) and selected concentrations of UDP-glucose orATP. Incubation was terminated by the addition of ice-cold 50 mMTris/HCl pH 7.4, 1 mM EDTA, 5 mM MgCl₂ and was followed immediately byfiltration under vacuum through Gelman A/E glass filters (Pall lifescience, An Arbor, Mich.) pre-soaked in binding buffer. The filters wererinsed twice before the addition of 5 ml of scintillation fluid(OpticFluor, Groninge, The Netherlands). Receptor-bound radioactivitywas measured using liquid scintillation analyzer Tricarb 2200 CA fromParckard. All assays were performed in triplicate. [³H]UDP-glucosebinding assays were also performed in isolated EGFP(+) and EGFP(−)cells. Membranes were incubated with a saturating concentration of[³H]UDP-glucose for 3 hours at 22° C. The non-specific [³H]UDP-glucosebinding was determined in the presence of 10 μM unlabeled UDP-glucose.The specificity of [³H]UDP-glucose binding was demonstrated in thepresence of a saturating concentration of ATP (10 μM). Incubations werestopped by the addition of ice-cold buffer and receptor-boundradioactivity was determined as described above. The equilibriumdissociation constant (Kd) and the capacity of binding indose-displacement studies were calculated using a scatchard plot and areexpressed as the mean±SD. Statistical analysis were performed using theunpaired Student t-test.

Immunoblotting

Proteins were run on NuPAGE Novex bis/tris 4-12% gels (Invitrogen) andtransferred to nitrocellulose membranes (Bio-Rad). After blocking (5%BSA in TBS 0.1% Tween 20 for 1 h), membranes were incubated overnightwith the primary antibody, as indicated in the text. After 3 washes inTBS 0.1% Tween 20, horseradish peroxidase-conjugated secondaryantibodies diluted 1:10,000 in TBS 0.1% Tween 20 were applied for 1 h atRT. Membranes were assayed with Western Lightning Chemiluminescencereagent (Perkin Elmer Life Sciences, Waltham, Mass., USA) and Kodakimaging films.

Immunofluorescence

Mice were anesthetized using pentobarbital sodium (50 mg/kg body, ip).The left kidney was perfused through the renal artery with PBS (0.9%NaCl in 10 mM phosphate buffer, pH 7.4), followed byparaformaldehyde-lysine-periodate fixative (PLP; 4% paraformaldehyde, 75mM lysine-HCl, 10 mM sodium periodate, and 0.15 M sucrose, in 37.5 mMsodium phosphate) for 10 min at a constant rate of 3.5 ml/min. Kidneyswere further fixed by immersion in PLP for 4 h at room temperature andsubsequently overnight at 4° C. After extensive washes in PBS,cryo-protection was performed in PBS containing 0.9 M (30% wt/vol)sucrose overnight at 4° C. Prior to cryo-sectioning, tissues wereembedded in Tissue-Tek OCT compound 4583 (Sakura Finetek USA, Torrance,Calif.) and frozen at −20° C. Sections (4-10 μm) were cut on a LeicaCM3050-S cryostat (Leica Microsystems, Bannockburn, Ill.) and stored at4° C. until use (37, 72). Sections were rehydrated in PBS and antigenretrieval techniques were performed by microwave heating in alkalinesolution (10 mM Tris buffer, 1 mM EDTA, pH 9.0) 3 times for 1 min, with5 min interval and then cooled down to room temperature. Sections werethen treated with 1% (wt/vol) SDS for 4 min (76). After washes in PBS,and incubation for 20 min in 1% (wt/vol) BSA in PBS the sections wereincubated for 60 min or overnight at 4° C. with the primary antibodydiluted in PBS containing 1% BSA. The secondary antibody was applied for1 h at room temperature and slides were mounted in Vectashield H1200medium containing 4,6-diamidino-2-phenylindole (DAPI) (VectorLaboratories, Burlingame, Calif.). Digital images were acquired using aNikon 90i epifluorescence microscope (Nikon Instruments, Melville,N.Y.). Images were analyzed using Volocity version 6.2.1image-processing software (Perkin Elmer), and imported into AdobePhotoshop software as TIFF files and the levels command was applied tothe entire field of view to better represent the raw data visualizedunder the microscope.

MDCK-C11 cells grown to confluence on filter (Corning) were biotinylatedas described above and were then fixed for 30 min in 4% paraformaldehyde(Electron Microscopy Sciences, Hatfield, Pa.). Cells were washed threetimes with PBS and treated with 1% SDS for 4 min for antigen retrieval.After several washes in PBS and blocking of the proteins with 1% BSA for30 min, cells were incubated for 1 h with anti-P2Y14 antibody diluted1:200 in PBS containing 1% BSA. Donkey anti-rabbit Cy3-conjugatedantibody (1:800) and FITC-conjugated streptavidin (1:1000) were appliedfor 40 min at RT. After three washes with PBS, cells were mounted withVectashield (Vector Laboratories) and visualized with a Zeiss Radiance2000 laser scanning confocal microscope (Zeiss Laboratories) usingLaserSharp 2000 version 4.1 software. Z-series (0.25 μm interval) weretaken for X-Z side view representations.

Statistical Analysis

The effects of treatments between two groups were determined by unpairedStudent's t-test when appropriated. Comparisons between multigroups weredetermined by one-way ANOVA followed by a post-hoc t-test. All testswere two-tailed, and P<0.05 was considered as statistically significant.

Results

P2 Receptor mRNA Expression in Intercalated Cells

P2X and P2Y receptor mRNA expression was first analyzed by conventionalRT-PCR in IC-enriched EGFP(+) cells isolated by FACS from the kidneys ofB1-EGFP mice as well as in whole kidney. In these mice, EGFP expressionis driven by the promoter of the V-ATPase B1 subunit, and occursspecifically in type A intercalated cells (A-ICs), type B intercalatedcells (B-ICs), and in the connecting tubules (CNT) (35). It waspreviously shown that a highly enriched EGFP(+) cell preparation couldbe generated that is depleted from all other cell types after FACSisolation (36, 37). As shown in FIG. 1, while transcripts specific forall P2 receptors, except P2Y4, were detected in whole kidney extracts(bottom panel), this number was narrowed down to 3 P2X (P2X₁, P2X₄ andP2X₅) and 3 P2Y (P2Y₂, p2y5 and P2Y₁₄) receptors in EGFP(+) cells (FIG.1, top panel).

The regional separation of B-ICs and CNT cells relative to A-ICs wasexploited. B—ICs and CNT cells are located exclusively in the renalcortex and to a lesser extent in the outer stripe of the outer medulla(OS), and A-ICs are located in most kidney regions (except the tip ofthe papilla) (38, 39). A mixed EGFP(+) cell population containing A-ICs,B—ICs and CNT cells was isolated from the kidney cortex and was comparedwith a “pure” A-IC population isolated from the inner stripe of theouter medulla (IS) and inner medulla. Quantitative PCR showed a markedenrichment of mRNA transcripts specific for the V-ATPase B1 subunit (amarker of A-ICs, B—ICs and CNT cells) (40-42), AE1 (a marker of A-ICs)(43) and pendrin (a marker of B-ICs) (44, 45) in EGFP(+) cells comparedto EGFP(−) cells isolated from the same regions (FIG. 2A). As expected,an increase in AE1 mRNA and a decrease in pendrin mRNA were detected inEGFP(+) cells isolated from the medulla versus the cortex, respectively,demonstrating the enrichment of A-ICs in the medullary EGFP(+) cellsversus cortical EGFP(+) cell populations. Quantitative PCR showedsignificantly higher expression levels for P2X₄, P2Y₂ and P2Y₁₄ inEGFP(+) cells isolated from the medulla compared to the cortex (FIG.2B). P2X₅ and p2y5 expression appeared to be similar in both regions andP2Y₁ was detectable only in cortical EGFP(+) cells, suggesting thatA-ICs most probably do not express P2Y₁. The very high P2Y₁₄ enrichment(by more than 20 fold) that was measured in medullary EGFP(+) cellscompared to cortical EGFP(+) cells prompted further characterization ofthe role of this receptor in ICs.

P2Y14 is Exclusively Expressed in Intercalated Cells

Double-immunofluorescence labeling of kidney sections for P2Y₁₄(visualized as green) and the V-ATPase B1 subunit (visualized as red)showed specific expression of P2Y₁₄ in A-ICs and B-ICs in the cortex,while no P2Y₁₄ was detected in the distal and connecting tubules (FIG.3A). In the medulla, P2Y₁₄ was detected only in A-ICs (FIG. 3B). P2Y₁₄was expressed in the apical region of both A-ICs and B-ICs, as comparedto the V-ATPase B1 subunit, which is apical in A-ICs, but basolateral orbi-polar in B-ICs (40). Pre-incubating the P2Y₁₄ antibody with itsimmunizing peptide abolished the P2Y₁₄ staining in both the cortex (FIG.3C) and medulla (FIG. 3D). P2Y₁₄ was also detected in occasional immunecells, which remained attached to the blood vessel walls (data notshown), consistent with its previously described localization incirculating immune cells (28-30). Immunoblotting of EGFP(+) cellextracts with the P2Y₁₄ antibody showed a predominant 50-KDa band and aweaker 40-kDa band (FIG. 4A). A radiolabeled UDP-glucose binding assaywas then performed using total membranes separated from FACS isolatedEGFP(+) cells and EGFP(−) cells (FIG. 4B). The [³H]UDP-glucose bindingmeasured in EGFP(+) cells was displaced with a saturating concentrationof cold UDP-glucose [10⁻⁵M], but not with ATP [10⁻⁵M], showingUDP-glucose-specific binding. In contrast, in EGFP(−) cells noUDP-glucose-specific binding was measured. Altogether, these data showthat ICs are the only renal epithelial cells that express P2Y₁₄.

UDP-Glucose Enhances ERK1/2 Phosphorylation in ICs

In human embryonic kidney (HEK) 293 cells stably transfected with P2Y₁₄,UDP-glucose stimulates the mitogen activated protein kinase (MAPK)pathway to phosphorylate extracellular signal-regulated kinase (ERK)1/2(46). It was, thus, tested whether this pathway was activated byUDP-glucose in ICs. FACS-isolated EGFP(+) ICs were treated in vitro with100 μM UDP-glucose for 15 min and ERK1/2 phosphorylation was measured byWB. FIGS. 5A-5B show a significant increase in ERK1/2 phosphorylationfollowing UDP-glucose treatment in EGFP(+) cells (left panel). Noincrease in ERK-phosphorylation was seen in EGFP(−) cells (FIG. 5A,right panel), confirming the specificity of P2Y₁₄ activation byUDP-glucose in ICs. Quantification showed a significant increase inERK1/2 phosphorylation in EGFP(+) cells but not in EGFP(−) cells (FIG.5B).

Characterization of the P2Y₁₄ Signaling Pathway in the Renal EpithelialCell Line MDCK-C11

The Madin-Darby Canine Kidney (MDCK) subclone C11 (MDCK-C11) was firstused as a renal epithelial cell model for the initial identification andcharacterization of the P2Y₁₄ signaling pathway. MDCK-C11 cells werepreviously shown to possess some characteristics of ICs, including Cl⁻and H⁺ secretion and the activation of H⁺ secretion by cAMP (47). Inagreement with this notion, RT-PCR analysis showed expression of markersof ICs, including the V-ATPase B1 and a4 subunits in these cells (FIG.6A). AQP2, a marker of collecting duct principal cells, was notdetected, supporting their non-principal cell phenotype (47). MDCK-C11cells did not express AE1 and thus do not retain all characteristics ofA-ICs. However, expression in MDCK-C11 cells of the V-ATPase B1 and Asubunits was confirmed at the protein level by western blotting, intotal cell lysates and in a biotinylated plasma membrane proteinfraction (FIG. 6B). The same membranes were re-blotted for actin. Theabsence of actin staining in the cell surface preparation showed theabsence of biotin contamination of intracellular proteins in thissample. RT-PCR analysis showed transcripts specific for P2Y₁, P2Y₄,p2y10, P2Y₁₂ and P2Y₁₄ (FIG. 6C). With the exception of P2X₂ all otherP2X receptors were also detected. Western blotting analysis showedexpression of P2Y₁₄ protein in total cell lysates and at the cellsurface (FIG. 6D). In agreement with a previous report in glioma C6cells (48), a more diffuse band at higher molecular weight was detectedin the plasma membrane compared to the 50 kDa band in total celllysates, indicating that only the glycosylated receptor can reach thecell surface. Glycosylation of P2Y₁₄ was confirmed by PNGaseF treatment,which resulted in the appearance of the deglycosylated form of P2Y₁₄ ataround 45 KDa in total cell lysates and in cell surface proteinextracts. Confocal microscopy showed P2Y₁₄ localization in the apicalplasma membrane (visualized as red), which was biotinylated and labeledwith biotin (visualized as green) in MDCK-C11 cells grown on filters(FIG. 6E). P2Y₁₄ is also expressed in the sub-apical region of thecells. The functionality of the receptor in MDCK-C11 cells was theninvestigated by using a radiolabeled UDP-glucose binding assay. Thespecificity of UDP-glucose binding was analyzed by dose-displacement of[³H]UDP-glucose in the presence of unlabeled UDP-glucose or ATP. Asshown in FIG. 6F, the addition of increasing concentrations of unlabeledUDP-glucose diminished [³H]UDP-glucose binding in a dose-dependentmanner. Concentration of ATP of up to 10 μM had no significantinhibitory effect on the [³H]UDP-glucose binding. Scatchard analysis ofthe binding shows only one class of binding site with an affinity of11.5±1.3 nM and a maximal binding capacity of 16.0±0.2 pmol/mg ofprotein. These values are very similar to the values obtained in HEK-293cells expressing P2Y₁₄ (previously known as KIAA0001) (16).

UDP-Glucose Increases ERK1/2 Phosphorylation Through P2Y₁₄ Activation inMDCK-C11 Cells.

As shown in FIG. 7A (upper panel), UDP-glucose (100 μM) induced asignificant increase in ERK1/2 phosphorylation in MDCK-C11 cells. Thiswas prevented by pre-treatment with the P2Y14 antagonist,4-((piperidin-4-yl)-phenyl)-(7-(4-(trifluoromethyl)-phenyl)-2-naphthoicacid (PPTN, 10 μM). Quantification of the ratio of p-ERK1/2 to totalERK1/2 showed a significant increase in ERK1/2 phosphorylation followingUDP-glucose treatment, which was abolished in the presence of PPTN (FIG.7B, bottom panel). An increase in the phosphorylation of other MAPKtargets such as p38 and JNK/SAPK (data not shown) was not detected,consistent with a previous report (46).

P2Y14 Activation Up-Regulates Pro-Inflammatory Chemokine mRNAs ThroughERK-Phosphorylation

The effects of P2Y₁₄ activation with UDP-glucose on mRNA expression ofseveral pro-inflammatory chemokines and cytokines were assessed here,both in MDCK-C11 cells (FIGS. 8A-8C) and renal EGFP(+) cells isolated byFACS from B1-EGFP mice (see below). As shown in FIG. 8A, MDCK-C11 cellsincreased IL-8 and CCL-2 mRNA expression following 4 h treatment with100 μM UDP-glucose. IL-8 and CCL-2 (also known as MCP-1) are well-knownchemo-attractants for neutrophils and monocytes, respectively. There wasno detectable increase of other pro-inflammatory mediators such as CCL4,CCL5, IL1β or TNFα. To determine the contribution of ERK-phosphorylationin cytokine production, ERK1/2-phosphorylation was inhibited by usingthe MEK inhibitor PD98059 (51). The treatment abolished the UDP-glucoseinduced up regulation of IL-8 and CCL-2 (FIG. 8B). In addition,pretreatment of MDCK-C11 cells with the P2Y₁₄ antagonist PPTN (50) alsoabolished the increase in IL8 and CCl2 mRNA expression induced byUDP-glucose (FIG. 8C).

To determine whether P2Y₁₄ activation induces the upregulation ofpro-inflammatory mRNAs in ICs in vivo, B1-EGFP mice were injectedthrough the tail vein with either a saline solution (sham) or a solutioncontaining 100 μM UDP-glucose. Kidneys were harvested 4 h later andprocessed for FACS isolation of EGFP(+) cells, mRNA extraction andreal-time PCR to measure pro-inflammatory chemokine and cytokineexpression. To avoid contamination of blood immune cells, which alsoexpress P2Y₁₄, the blood was flushed out of the kidneys by perfusingmice with PBS through the cardiac left ventricle. In addition, maximumpurity (>95%) of EGFP(+) cells was obtained by further restricting thesorting parameters to isolate bright EGFP(+) cells. Cytospin smears ofEGFP(+) cells immunostained for CD45 (a marker of leukocytes) did notshow any contamination of the samples with leukocytes (data not shown).It was thus clear that any changes in pro-inflammatory mediatorexpression following P2Y₁₄ activation with UDP-glucose were attributedto the presence of the receptor in ICs uniquely. As shown in FIG. 9, theneutrophil chemo-attractants, CXCL1 (KC) and CXCL2 (MIP-2α) (bothhomologues of the murine IL-8) had significantly higher expressionlevels following UDP-glucose treatment. A significant increase was alsoobserved for the monocyte chemo-attractant CCL2 (MCP-1) and CCL3(MIP-1α). No significant changes were observed for CCL4, CCL5, TNFα,IL1β, and IL6 expression. These results show that ICs producepro-inflammatory mediators in vivo following activation by apro-inflammatory agonist, and that this process can be efficientlypromoted through UDP-glucose/P2Y₁₄ signaling.

UDP-Glucose Activation Induces Neutrophil Infiltration into the KidneyMedulla

The pathophysiological relevance of P2Y₁₄ expression in ICs was assessedby measuring the infiltration of immune cells into the kidney using flowcytometry in mice that were challenged with an injection of UDP-glucose.To address the spatial distribution of immune cells in the kidney, thekidney was separated into cortex and medulla. B1-EGFP mice werechallenged with a tail-vein injection of either 100 μM UDP-glucose(treated) or saline (sham), as discussed above. Mice were perfused 48 hlater through the cardiac left ventricle with PBS to flush the blood outof the kidney vessels and hence measure only tissue infiltrated immunecells. This approach identified a significant and selective accumulationof neutrophils in the kidney medulla (FIG. 10A). This observation isconsistent with the upregulation of neutrophil chemo-attractantsobserved in EGFP(+) cells after UDP-glucose activation.Anti-inflammatory (Ly6C low) monocytes also slightly decreased in themedulla, otherwise no significant changes in other immune cell countswere observed. In the kidney cortex, the numbers of most cell typesremained unchanged upon UDP-glucose treatment, except for the Ly6C lowmonocyte population, which decreased slightly (FIG. 10B). No apparentchanges in the number of infiltrated immune cells were observed in thecortex and medulla at earlier time points (12 and 24 hours) followingUDP-glucose injection (data not shown).

The UDP-Glucose Induced Neutrophil Infiltration Occurs Primarily in theRenal Medulla

Kidney sections from control and UDP-glucose treated mice were stainedfor P2Y₁₄ (visualized as green) and for Ly6G, a neutrophil marker(visualized as red). Mosaic images were captured and neutrophilsidentified based on their positive labeling for Ly6G and theirpoly-nucleated phenotype (white circles) (FIGS. 11A-11B). Moreneutrophils were visible in the medulla of UDP-glucose treated mice(FIG. 11B) compared to sham-treated control mice (FIG. 11A). Highermagnification images show the presence of neutrophils in the renalmedulla in UDP-glucose treated mice (FIGS. 11D, 11D′, 11D″) but not incontrols (FIGS. 11C, 11C′, 11C″).

Discussion

In this study, the P2 receptor profile of renal intercalated cells wascharacterized, and it was shown that they express high levels of thepro-inflammatory P2Y₁₄ receptor. In addition, the data provided hereindemonstrate that P2Y₁₄ activation by UDP-glucose in ICs induces ERK1/2phosphorylation followed by an inflammatory response mediated bychemokine upregulation and neutrophil recruitment into the kidneymedulla. This study, therefore, identifies ICs as both sensors andmediators of sterile inflammation in the kidney.

While numerous P2 receptors were detected in mRNA samples isolated fromthe entire kidney, only 6 (P2X₁, P2X₄, P2X₅, P2Y₂, p2y5 and P2Y₁₄)receptors were found in ICs. The expression of P2Y₂ in these cells is inagreement with a previous pharmacological study showing functional P2Y₂in the apical membrane of ICs in rabbit CCDs (52). P2Y₂ is traditionallyviewed as a regulator of collecting duct principal cells, but these datafurther support its participation in IC function. Interestingly, whileP2X₇ and P2X₆ have been described in collecting ducts (53, 54), thesereceptors were not detected in EGFP(+) cells, illustrating theimportance of conducting cell-specific gene expression analysis. Amongthe receptors that were detected in ICs, P2X₄ is regulated byextracellular pH (55) and it will be interesting to determine its rolein the acidifying function of ICs.

The role of ICs in the defense against ascending pathogens has recentlyemerged by the discovery that they express the antimicrobial agent RNAse7 and its modulator, the ribonuclease inhibitor (RI) (56-58). It wasproposed that ICs are involved in the maintenance of luminal sterilityin the kidney. Ascending pathogens induce renal epithelial cell damage(59-61). Uropathogenic Escherichia coli can adhere to the apical surfaceof medullary ICs, where it activates TLR4-dependent and independentsignaling pathways (62). Multiple studies have characterized Toll-likereceptors (TLR) as pathogen recognition and inflammation mediators inthe kidney (62-64). In contrast to infection-induced inflammation, themolecular mechanisms that stimulate inflammation secondary to kidneydiseases, nephrotoxicity and renal transplantation among others arepoorly understood. Infection and ischemic kidney injury stimulate apotent inflammatory response including a rapid infiltration ofneutrophils into the affected tissue (65). This present study providesevidence for a parallel non-TLR mediated inflammatory pathway determinedby ICs. Without wishing to be bound by theory, the UDP-glucose releasedfrom damaged cells activates P2Y₁₄ receptor in ICs to initiate aninflammatory response via the production of pro-inflammatory chemokines,which then recruit neutrophils to damaged and/or infected areas. Withoutwishing to be bound by theory, P2Y₁₄ receptors act as danger sensors inkidney ICs. Intriguingly it was found that P2Y₁₄ mRNA expression is 20times higher in medullary ICs compared to cortical ICs, despite the factthat immunostaining detected abundant protein in both cortical andmedullary ICs. The renal medulla is the primary site of exposure tourinary ascending pathogens, which induce renal epithelial cell damage(59-61). The large amount of P2Y₁₄ mRNA seen in this region could beexplained by the necessity to readily synthesize a functional proteinimmediately following infection, in order to induce a rapid inflammatoryresponse. It could also reflect a higher level of mRNA expression inA-type ICs, which are enriched in the isolated medullary preparationcompared to the cortical EGFP(+) cells.

P2Y₁₄ activates the MAPK pathway, which regulates the stability of IL-8mRNA as opposed to LPS, which transcriptionally activates the IL-8 genevia the NF-kB pathway (66). In agreement with this notion, it was shownhere that UDP-glucose administered both in vitro and in vivo induces apotent inflammatory response in ICs by up-regulating pro-inflammatorychemokine expression through MAPK activation and subsequent recruitmentof neutrophils into the renal medulla. This would suggest that P2Y₁₄(MAPK activation) and LPS (mainly NF-kB activation) act in parallel toincrease IL-8 secretion and neutrophil recruitment.

Cytokines are released by leukocytes and renal tubular cells (67), andthey are important contributors to the initiation of inflammation in theinjured kidney. Pro-inflammatory cytokines such as TNFα, IL6 and IL1βinduce chemokines through complementary activation, and the NF-kB andTLR related pathways. In this study it was shown that the increase inneutrophil and monocyte chemo-attractants is not accompanied by anincrease of IL1β, TNFα or IL6, indicating that UDP-glucose can itselfact as a pro-inflammatory mediator bypassing the cytokine effects.Without wishing to be bound by theory, by their ability to producechemokines, ICs act as immune defense cells by creating a chemotaxicgradient favorable to neutrophil recruitment.

The increase in the amount of infiltrated neutrophils that was observedin the kidney medulla following UDP-glucose administration is inquantitative agreement with previous studies showing a 3-fold increasein renal neutrophil content after bilateral ischemia-reperfusion (67),and a 4-fold increase in the mouse uterus after UDP-glucoseadministration (33). In addition, significant neutrophil infiltrationwas found after 48 hours, a time course identical to the infiltrationobserved in the mouse uterus (33). Interestingly, a decrease in Ly6C lowmonocyte content following UDP-glucose administration was also observed.These cells have anti-inflammatory characteristics (68-70). Indeed arecent study suggested a role for Ly6C low monocytes as neutrophilrecruiters in the presence of a “danger signal” in kidney blood vessels(71). This study showed that following TLR7 activation, the kidneyendothelium retains crawling monocytes within the capillaries. Withoutwishing to be bound by theory, under basal conditions some of thecrawling monocytes would eventually penetrate into the tissue whereaswhen a “danger signal” is sensed, the capillaries would retain themonocytes in order to recruit neutrophils, in which case the tissueinfiltrated Ly6C low monocyte counts would decrease as indeed observedin this study.

In conclusion, the data provided herein show that ICs are DAMP sensorsthat mediate the recruitment of pro-inflammatory neutrophils to thekidney via activation of P2Y14. Almost all kidney diseases trigger astrong inflammatory response that can ultimately lead to kidney failure.The study presented herein, therefore, identifies P2Y14 as a usefultherapeutic target for the, detection, prevention, or treatment ofsterile inflammation in the kidney.

TABLE 1 GeneBank Gene accession Forward primer (5′-3′)Reverse primer (5′-3′) Mouse NM_008771 AGGGTCTCAAACACCCACAGAGCTGTTCCAACCCACAGAG P2rx1 (SEQ ID NO: 1) (SEQ ID NO: 2) Mouse NM_153400TTTGGCCCAACTTTGATCTC CCCAGAGCAAGATGCCTATC P2rx2 (SEQ ID NO: 3)(SEQ ID NO: 4) Mouse NM_145526 AGCCTCTTCTGGGACATCAA GTTAGGGATGGCGCTGAGTAP2rx3 (SEQ ID NO: 5) (SEQ ID NO: 6) Mouse NM_011026 AGCAGCTCTGTCCAAGCACTTCCGAGGACAACTTCTCTGG P2rx4 (SEQ ID NO: 7) (SEQ ID NO: 8) Mouse NM_033321AAGGGGAGAGCAAACACTCA CACCAACCAAACAGCAAGTG P2rx5 (SEQ ID NO: 9)(SEQ ID NO: 10) Mouse NM_011028 GACGATCCTGGTCCAAGTGTACCAGGAACTCCAAGGGTTT P2rx6 (SEQ ID NO: 11) (SEQ ID NO: 12) MouseNM_011027 ACGCTTTCTTCAGCAGCAAT GCTGCTCTCAGTTCTGACCA P2rx7(SEQ ID NO: 13) (SEQ ID NO: 14) Mouse NM_008772 GCATTTTGAGCCTTCTCAGGCTCCCTCCAGCCAAACAATA P2ry1 (SEQ ID NO: 15) (SEQ ID NO: 16) MouseNM_008773 CAGCACAAACCATGCTGACT ACAAGGGACCTCCTGTCCTT P2ry2(SEQ ID NO: 17) (SEQ ID NO: 18) Mouse NM_020621 CAGACCAAAGAGCACGAACAGCTGGAACAGCAATGGAACT P2ry4 (SEQ ID NO: 19) (SEQ ID NO: 20) MouseBC069991 GGGCACTGAGAATTTTATCCA GAGCAGTCCCAGTGGCTTAG P2ry5(SEQ ID NO: 21) (SEQ ID NO: 22) Mouse NM_183168 TAGGCCCTGGAATAGCAATGTCTTGGCAAATGGATGTGAA P2ry6 (SEQ ID NO: 23) (SEQ ID NO: 24) MouseNM_172435 CAGAACCCCCATCTTCTCAA GTGGTCCCTTCCTCTTCCTT P2ry10(SEQ ID NO: 25) (SEQ ID NO: 26) Mouse NM_027571 AAAATGCCTGCTGCTTGAATTGAAGAAATTCCAACAAAACGA P2ry12 (SEQ ID NO: 27) (SEQ ID NO: 28) MouseNM_028808 AAGCCACAGAGGCAAGAGAA CCTGGAGTAAGGGACAGCAA P2ry13(SEQ ID NO: 29) (SEQ ID NO: 30) Mouse NM_133200 CCATGCAAAATGGAAGTCTGCGGAAAGACTGGGTGTCTTC P2ry14 (SEQ ID NO: 31) (SEQ ID NO: 32) MouseNM_008084.2 GCACAGTCAAGGCCGAGAAT GCCTTCTCCATGGTGGTGAA Gapdh(SEQ ID NO: 33) (SEQ ID NO: 34) Mouse NM_011867.3 TTAGCAATGTTCGGATGTGCGGCCAGCCTAACAGAGACAG Slc26v1 (SEQ ID NO: 35) (SEQ ID NO: 36) MouseNM_134157.2 ACACGGCGCTCTAAATCAGT CCACCCACCTACACCAAAAG Atp6v1b1(SEQ ID NO: 37) (SEQ ID NO: 38) Mouse NM_011403.2 TAGAAATGAGGGCAGGGATGGCAAAACCTATTCCAA Slc4v1 (SEQ ID NO: 39) (SEQ ID NO: 40) MouseNM_008176.3 TGTTGTGCGAAAAGAAGTGC CGAGACGAGACCAGGAGAAA Cxcl1(SEQ ID NO: 41) (SEQ ID NO: 42) Mouse NM_009140.2 CGGTCAAAAAGTTTGCCTTGTCCAGGTCAGTTAGCCTTGC Cxcl2 (SEQ ID NO: 43) (SEQ ID NO: 44) Mouse TnfNM_013693.2 CCACCACGCTCTTCTGTCTAC AGGGTCTGGGCCATAGAACT (SEQ ID NO: 45)(SEQ ID NO: 46) Mouse NM_011333.3 CAAGAAGGAATGGGTCCAGAAAGGCATCACAGTCCGAGTC Cc12 (SEQ ID NO: 47) (SEQ ID NO: 48) MouseNM_011337.2 TAGCCACATCGAGGGACTCT ACCAACTGGGAGGGAGATG Cc13(SEQ ID NO: 49) (SEQ ID NO: 50) Mouse NM_013652.2 GATTTCCTGCCCCTCTTCTTGGGAGACACGCGTCCTATAA Cc14 (SEQ ID NO: 51) (SEQ ID NO: 52) MouseNM_013653.3 GTGCCCACGTCAAGGAGTAT CCACTTCTTCTCTGGGTTGG Cc15(SEQ ID NO: 53) (SEQ ID NO: 54) Mouse NM_008361.3 GGGCCTCAAAGGAAAGAATCTACCAGTTGGGGAACTCTGC I11h (SEQ ID NO: 55) (SEQ ID NO: 56) Mouse I16NM_031168.1 GTGGCTAAGGACCAAGACCA ACCACAGTGAGGAATGTCCA (SEQ ID NO: 57)(SEQ ID NO: 58) Canine XM_548344.2 TTCGCTTTGACATTCTCGTGCATTTGCTCCGCATACTTGA P2RX1 (SEQ ID NO: 59) (SEQ ID NO: 60) CanineXM_534633.3 ACACTCTCCATCCTGCTGCT TGAGAGGAAGTCAGGGGAGA P2RX2(SEQ ID NO: 61) (SEQ ID NO: 62) Canine XM_540614.1 GACTGTCCTCTGCGACATCATTAGTGGCCGATGGAGTAGG P2RX3 (SEQ ID NO: 63) (SEQ ID NO: 64) CanineXM_003639907.1 CCACAGTCCTCATCAGAGCA GCCAGAGGTCACCTGAACAT P2RX4(SEQ ID NO: 65) (SEQ ID NO: 66) Canine XM_003639268.1GCCAGCCACTTCTCTTTGTC CAAATCCCACTCAGCCATTT P2RX5 (SEQ ID NO: 67)(SEQ ID NO: 68) Canine XM_543562.1 CTGGGTGTGATCACCTTCCTGAAGCTGGCTTTGTCTGCTC P2RX6 (SEQ ID NO: 69) (SEQ ID NO: 70) CanineNM_001113456.1 CCCACATTAGGATGGTGGAC CAGCCTGGACAAGTCTGTGA P2RX7(SEQ ID NO: 71) (SEQ ID NO: 72) Canine NM_001193673.1GCTTGTGAAGAGGCAGGAAC TCACTGGATCCACAGTCCAA P2RY1 (SEQ ID NO: 73)(SEQ ID NO: 74) Canine XM_542321.2 CGTCAACGTGGCTTACAAGAAATCCTCACTGCTGGTGGAC P2RY2 (SEQ ID NO: 75) (SEQ ID NO: 76) CanineXM_003640257.1 ATGTGAGCTCTGGCAGCTTT GGAAGCCACAGTGAGTGGAT P2RY4(SEQ ID NO: 77) (SEQ ID NO: 78) Canine XM_542320.1 CTTCCTGCCCTTCCATGTTAGGCGGAACTTCTTCTGAGTG P2RY6 (SEQ ID NO: 79) (SEQ ID NO: 80) CanineXM_549100.2 GCACTGCGGATGGTTTTTAT CAGTGTGCTTTGGACAATGG P2RY10(SEQ ID NO: 81) (SEQ ID NO: 82) Canine NM_001003365.1CAAGAGGCGTAGGCAAAGTC GTAGGGAATGCGTGCAAAAT P2RY12 (SEQ ID NO: 83)(SEQ ID NO: 84) Canine XM_542838.2 CTCATTACAGCTGCCGATCATCTAAAGGGCTGGCATAGGA P2RY14 (SEQ ID NO: 85) (SEQ ID NO: 86) CanineNM_001003142.1 GCCCTCAATGACCACTTTGT TCCTTGGAGGCCATGTAGAC GAPDH(SEQ ID NO: 87) (SEQ ID NO: 88) Canine XM_540382.3 AACTCCGAGCTTCCAGTCAATCTCACTCCAACGACATCCA SLC26A4 (SEQ ID NO: 89) (SEQ ID NO: 90) CanineXM_531858.3 CAAATCTACCCTCCGGTCAA GGTTGATGAAGCTCCTCTCG ATP6V1B1(SEQ ID NO: 91) (SEQ ID NO: 92) Canine XM_539895.3 CAGCCTTGTCTTCAACGTCACTTGAGGTCGGTTCCCCTAT ATP6V0A4 (SEQ ID NO: 93) (SEQ ID NO: 94) CanineNM_001048031.1 TCATCCTCACTGTGCCTCTG CTCTGAGGCTCACACCTTCC SLC4A1(SEQ ID NO: 95) (SEQ ID NO: 96) Canine XM_543678.3 GGGCTCCCTCCTCTACAACTGCAGCTCCACTGACTGTCG AQP2 (SEQ ID NO: 97) (SEQ ID NO: 98) Canine IL8NM_001003200.1 TCAATTGAACCGCAATCCTA TGCTTGTCGAGTTTTTGCTC (SEQ ID NO: 99)(SEQ ID NO: 100) Canine NM_001003244.4 TCATCTTCTCGAACCCCAAGCTGGTTGTCTGTCAGCTCCA TNF (SEQ ID NO: 101) (SEQ ID NO: 102) CanineNM_001003297.1 CAAGAAAAGCCAAACCCAAA GAGGGCATTTAGGGAAGGTT CCL2(SEQ ID NO: 103) (SEQ ID NO: 104) Canine NM_001005251.1CAAGCCCGGTATTATCTTCG AGGCTTTCAGCTTCAGATCG CCL3 (SEQ ID NO: 105)(SEQ ID NO: 106) Canine NM_001005250.1 CTTTGAGACCAGCAGCCTCTCAGTTCAGTTCCAGATCATCCA CCL4 (SEQ ID NO: 107) (SEQ ID NO: 108) CanineNM_001003010.2 GCTCTGCAGTCAGGAAGGAG GGCTGAGAGGATAGCTGTGG CCL5(SEQ ID NO: 109) (SEQ ID NO: 110) Canine NM_001037971.1CCTGTGTGATGAAGGATGGA TATATCCTGGCCACCTCTGG ILIB (SEQ ID NO: 111)(SEQ ID NO: 112) Canine IL6 NM_001003301.1 CTCGGCAAAATCTCTGCACTTGGAAGCATCCATCTTTTCC (SEQ ID NO: 113) (SEQ ID NO: 114)

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Example 2 P2Y14 is Expressed in Human Kidney Intercalated Cells.

It is shown that P2Y14 is expressed in intercalated cells (ICs) fromhuman kidney sections. ICs were identified by their positive labelingfor the proton pump, V-ATPase (FIG. 12).

Measurement of UDP-Glucose in Mouse Urine Samples.

UDP-glucose in urine samples from mice was measured. Urine samples werecollected in a paired manner in control mice, before and afterUDP-glucose injection through their tail vein. The goal was to firstdetermine the level of UDP-glucose in the urine of healthy mice, andthen to determine the threshold at which neutrophil infiltration occursin the kidney. A previously published protocol for the measurement ofUDP-glucose in lung excretions was used (Barrett et al. Molec.Pharmacol. 2013). The data obtained show a very low UDP-glucoseconcentration under control conditions (30 nM range), and a significantincrease (200 nM) after a single IV injection with a solution containingUDP-glucose.

UDP-Glucose Measurements in Human Urine Samples

UDP-glucose levels are measured in the urine samples from healthyindividuals, as well as from patients with AKI, CKD and sepsis.UDP-glucose levels are also measured in a longitudinal study thatexamines patients who are admitted in the intensive care unit. Urinesamples are collected every day in these patients, some of whom willdevelop AKI and some will not.

Murine Models of Kidney Disease

The novel pro-inflammatory function of ICs is explored in several murinedisease models.

It was confirmed that the murine studies could be applied to humanstudies by showing expression of P2Y14 in human ICs in addition tomurine ICs. It was also shown that very low levels of UDP-glucose werepresent in the urine of healthy mice, and that a single UDP-glucosechallenge induced a significant increase in urine concentration.

1. A method of treating renal inflammation comprising providing acomposition comprising a P2Y14 inhibitor in a therapeutically effectiveamount to treat or prevent renal inflammation.
 2. The method of claim 1,wherein the P2Y14 inhibitor is4-((piperidin-4-yl)-phenyl)-(7-(4-(trifluoromethyl)-phenyl)-2-naphthoicacid (PPTN).
 3. The method of claim 1, wherein the composition issuitable for delivery in an organ-specific delivery device.
 4. Themethod of claim 3, wherein the delivery device is a renal catheter. 5.The method of claim 1, wherein the composition is suitable for deliveryinto renal circulation.
 6. The method of claim 1, wherein the renalinflammation is associated with a condition selected from the groupconsisting of acute kidney injury, chronic renal failure,glomerulonephritis, heterologous nephrotoxic nephritis, animmune-related disease, nephropathy, pyelonephritis, and sclerosis ofthe glomerulus.
 7. The method of claim 6, wherein the renal inflammationis associated with acute kidney injury.
 8. The method of claim 1,wherein the renal inflammation is early stage.
 9. A method of treatingacute kidney injury comprising providing a composition comprising aP2Y14 inhibitor in a therapeutically effective amount to treat orprevent acute kidney injury.
 10. The method of claim 9, wherein theP2Y14 inhibitor is PPTN.
 11. The method of claim 9, wherein thecomposition is suitable for delivery in an organ-specific deliverydevice.
 12. The method of claim 11, wherein the delivery device is arenal catheter.
 13. The method of claim 9, wherein the composition issuitable for delivery into renal circulation.
 14. The method of claim 1,wherein the composition is formulated for administration by a routeselected from the group consisting of parenteral, intravenous,intralesional, and intratumoral.
 15. The method of claim 1, wherein thecomposition is formulated for administration by a mode selected from thegroup consisting of injection and infusion.
 16. The method of claim 9,wherein the composition is formulated for administration by a routeselected from the group consisting of parenteral, intravenous,intralesional, and intratumoral.
 17. The method of claim 9, wherein thecomposition is formulated for administration by a mode selected from thegroup consisting of injection and infusion.